Somatic cell nuclear transfer methods

ABSTRACT

The present invention provides methods for making reconstructed diploid human oocytes comprising the diploid genome of a human somatic cell, and also methods for making human nuclear transfer embryos, human embryonic stem cells, and human differentiated cells therefrom. The present invention also provides reconstructed human oocytes, human nuclear transfer embryos, human embryonic stem cells, and differentiated cells made using such methods, as well as compositions and kits useful in performing such methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/775,594 filed Sep. 11, 2015, now issued as U.S. Pat. No.9,896,699, which is a 35 USC § 371 National Stage application ofInternational Application No. PCT/US2014/029295 filed Mar. 14, 2014, nowexpired; which claims the benefit under 35 USC § 119(e) to U.S.Application Ser. No. 61/947,353 filed Mar. 3, 2014, U.S. ApplicationSerial No. 61/891,322 filed Oct. 15, 2013 and to U.S. Application Ser.No. 61/793,492 filed Mar. 15, 2013, all now expired. The disclosure ofeach of the prior applications is considered part of and is incorporatedby reference in the disclosure of this application.

COPYRIGHT

A portion of the disclosure of this patent document contains materialthat is subject to copyright protection. The copyright owner has noobjection to the facsimile reproduction by anyone of the patent documentor the patent disclosure as it appears in the Patent and TrademarkOffice patent file or records, but otherwise reserves all copyrightrights whatsoever.

INCORPORATION BY REFERENCE

For countries and territories that permit incorporation by reference,the text of all documents cited herein is hereby incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

The cloning of frogs from somatic cells demonstrated thatdifferentiation from the zygote into specialized cell types was areversible process. The transplantation of somatic nuclei intounfertilized mammalian oocytes resulted in the cloning of sheep, mice,cows and various other mammalian species.

The derivation of embryonic stem cells from human blastocysts broughtthe prospect of combining nuclear transfer and stem cell derivation togenerate cells and tissues for patients requiring replacement ofdiseased cells or tissue. This concept was realized in the mouse for thecorrection of immunodeficiency and of Parkinson's disease (Rideout etal. 2002 Cell 109(1): 17-27). Nuclear transfer stem cells were alsoderived from the rhesus monkey (Byrne et al., 2007 Nature 450 (7169):497-502). However, most previous attempts at human somatic cell nucleartransfer (SCNT) using human cells have resulted in the generation ofnuclear transfer embryos that consistently arrest at the late cleavagestages with karyotypic and transcriptional defects, prohibiting furtherdevelopment or stem cell derivation. Prior to the present invention, theonly SCNT methods that were shown to be effective in generating humanblastocyst stage embryos and stem cells derived therefrom were thosethat involved transferring a diploid human somatic cell genome into ahaploid human oocyte without removing the oocyte's genome. (See Noggleet al. 2011. Human oocytes reprogram somatic cells to a pluripotentstate. Nature 478(7367): 70-75. See also, U.S. Patent Application Pub.No.: US 2012/0129620). Such methods resulted in the generation ofembyros and stem cells that were triploid. Thus, prior to the presentinvention there remained a need in the art for a method of generating adiploid human nuclear transfer embryo capable of developing to theblastocyst stage and from which diploid human pluripotent stem cellscould be derived.

SUMMARY OF THE INVENTION

The present invention provides methods by which a human diploid embryocan be generated by somatic cell nuclear transfer. In some embodimentssuch methods involve transferring the diploid genome from a humansomatic cell into an enucleated human oocyte cell, resulting inreprogramming of the somatic cell genome to an embryonic state. Usingsuch methods the resulting reconstructed oocytes are able to develop tointo blastocyst stage embryos having an inner cell mass. Furthermore,the present invention provides methods by which such blastocyst stageembryos can be used to derive diploid pluripotent stem cells (embryonicstem cells) containing a somatic cell genome that has been reprogrammedto an embryonic state. The present invention also provides methods ofobtaining differentiated cells from such pluripotent stem cells. Theseand other aspects of the present invention are further describedthroughout the specification, claims, and drawings of this patentapplication.

In some aspects the present invention provides several importantimprovements over and above prior methods that result in improveddevelopment. These improvements include, but are not limited to, thefollowing. First, in some embodiments the methods of the presentinvention are designed to maintain plasma membrane integrity duringoocyte preparation and during cell /nuclear fusion. Second, in someembodiments the methods of the present invention are designed tominimize the negative consequences of compromised plasma membraneintegrity, should it occur, by using calcium-free media, calciumchelators, and phosphatase inhibitors, either alone or in combination.Third, in some embodiments the methods of the present invention aredesigned to enable rapid and efficient activation of human oocytesusing, for example using dual inhibition of both translation and meiotickinase activity. Fourth, in some embodiments the methods of the presentinvention are designed to maximize reprogramming by improvingreplication and segregation of the somatic cell genome in the activatedegg. Chromosome mis-segregation is frequent after somatic cell nucleartransfer. However, it is a discovery of the present invention thatagents applied during the first cell cycle, such as histone deacetylaseinhibitors and histone methylation inhibitors, can increase fidelity ofchromosome duplication and enable efficient development to theblastocyst stage. These and other aspects of the present invention aredescribed further below and thoughout the specification and claims ofthe present application.

It should be noted that while the methods of the present invention werecreated for, and shown to be effective in, human somatic cell nucleartransfer applications, the methods described herein may also be usefulin other applications, including for nuclear transfer using non-humansomatic cells and for nuclear transfer using non-somatic cells (such asoocyte nuclear transfer protocols). One of skill in the art will be ableto appreciate those aspects of the invention described herein that canbe applied equally to non-human somatic cell nuclear transfer methodsand to non-somatic cell nuclear transfer methods. Thus, in someembodiments of the invention the methods described herein can be appliedto non-human cells and to non-somatic cells. It should also be notedthat the different aspects of the methods described herein can beperformd in various different combinations and also that, in someembodiments, only particular aspects of the methods described hereinneed be performed. One of skill in the art will appreciate those aspectsof the present methods that can be practiced alone, or in combinationwith other methods, and all such methods are intended to fall within thescope of this invention. For example, in embodiments of the inventionthat comprise multiple separate method steps, the individual methodsteps can also be used in isolation, or in conjunction with othermethods. As described above, and throughout this specification, themethods of the present invention provide several improvements over andabove prior methods. In some embodiments all of the improvementsdescribed herein are used, while in other embodiments only one suchimprovement (e.g., the use of a calcium-free medium and/or calciumchelator during nuclear transfer, the use of a low amount of fusogenicagent, the use of a translation inhibitor, the use of a meiotic kinaseinhibitor, the use of a histone deacetylase inhibitor, etc.) need beused, and in yet other embodiments any combination of two or more ofsuch improvements may be used, as desired.

In one embodiment the present invention provides a method for producinga diploid human nuclear transfer embryo capable of developing into ablastocyst containing an inner cell mass and/or from which embryonicstem (ES) cells can be derived, the method comprising: obtaining adiploid nuclear genome from a postnatal human somatic cell, such as anadult human somatic cell.

In one embodiment the present invention provides a method for producinga diploid human nuclear transfer embryo capable of developing into ablastocyst containing an inner cell mass and/or from which ES cells canbe derived, wherein the method comprises transferring a diploid humansomatic cell nuclear genome into an enucleated mature human oocyte in amedium that is calcium-free, and/or contains a calcium chelator, and/orcontains a phosphatase inhibitor.

In one embodiment the present invention provides a method for producinga diploid human nuclear transfer embryo capable of developing into ablastocyst containing an inner cell mass and/or from which ES cells canbe derived, wherein the method comprises transferring a diploid humansomatic cell nuclear genome into an enucleated mature human oocyte usinga fusogenic agent, wherein the concentration of the fusogenic agent isselected so as to minimize cell membrane damage while still beingsufficient to induce cell fusion.

In one embodiment the present invention provides a method for producinga diploid human nuclear transfer embryo capable of developing into ablastocyst containing an inner cell mass and/or from which ES cells canbe derived, wherein the method comprises transferring a diploid humansomatic cell nuclear genome into an enucleated mature human oocyte usinga fusogenic agent, wherein the fusogenic agent is contacted with arestricted area of the somatic cell and/or the oocyte so as to minimizecell membrane damage while still inducing cell fusion.

In one embodiment the present invention provides a method for producinga diploid human nuclear transfer embryo capable of developing into ablastocyst containing an inner cell mass and/or from which ES cells canbe derived, the method comprising transferring a diploid human somaticcell nuclear genome into an enucleated mature human oocyte to form areconstructed oocyte and subsequently activating the reconstructedoocyte by contacting it with one or more of a calcium ionophore, aninhibitor of translation, and an inhibitor meiotic kinases.

In one embodiment the present invention provides a method for producinga diploid human nuclear transfer embryo capable of developing into ablastocyst containing an inner cell mass and/or from which ES cells canbe derived, the method comprising transferring a diploid human somaticcell nuclear genome into an enucleated mature human oocyte to form areconstructed oocyte and subsequently activating the reconstructedoocyte by contacting it with a calcium ionophore and an inhibitor oftranslation.

In one embodiment the present invention provides a method for producinga diploid human nuclear transfer embryo capable of developing into ablastocyst containing an inner cell mass and/or from which embryonicstem ES cells can be derived, the method comprising transferring adiploid human somatic cell nuclear genome into an enucleated maturehuman oocyte to form a reconstructed oocyte and subsequently activatingthe reconstructed oocyte by contacting it with a calcium ionophore, aninhibitor of translation and an inhibitor meiotic kinases.

In one embodiment the present invention provides a method for producinga diploid human nuclear transfer embryo capable of developing into ablastocyst containing an inner cell mass and/or from which ES cells canbe derived, the method comprising transferring a diploid human somaticcell nuclear genome into an enucleated mature human oocyte to form areconstructed oocyte, activating the reconstructed oocyte, andcontacting the reconstructed oocyte and/or embryo derived therefrom witha histone deacetylase inhibitor. In some such embodiments thereconstructed oocyte is contacted with the histone deacetylase inhibitorduring its first cell cycle.

In one embodiment the present invention provides a method for producinga diploid human nuclear transfer embryo capable of developing into ablastocyst containing an inner cell mass and/or from which embryonicstem ES cells can be derived, the method comprising transferring adiploid human somatic cell nuclear genome into an enucleated maturehuman oocyte to form a reconstructed oocyte, activating thereconstructed oocyte, and contacting the reconstructed oocyte and/orembryo derived therefrom with a histone methylation inhibitor. In somesuch embodiments the reconstructed oocyte is contacted with the histonemethylation inhibitor during its first cell cycle.

In one embodiment the present invention provides a method for producinga diploid human nuclear transfer embryo capable of developing into ablastocyst containing an inner cell mass, the method comprising: (a)obtaining a diploid nuclear genome from a postnatal human somatic cell,(b) obtaining an enucleated mature human oocyte, (c) transferring thediploid nuclear genome into the enucleated mature human oocyte to form areconstructed oocyte, wherein the transferring is performed in a mediumthat is calcium-free, and/or contains a calcium chelator, and/orcontains a phosphatase inhbitor, (d) subsequently contacting thereconstructed oocyte with a calcium ionophore, an inhibitor oftranslation, and an inhibitor meiotic kinases, to activate thereconstructed oocyte and promote entry into interphase, and (e)subsequently contacting the reconstructed oocyte with a histonedeacetylase inhibitor and/or a histone methylation inhibitor, therebyproducing a diploid human nuclear transfer embryo capable of developinginto a blastocyst containing an inner cell mass.

In another embodiment the present invention provides a method forproducing a diploid human nuclear transfer embryo capable of developinginto a blastocyst containing an inner cell mass, the method comprising:(a) obtaining a diploid human somatic cell nuclear genome, (b) obtainingan enucleated mature human oocyte, (c) transferring the diploid humansomatic cell nuclear genome into the enucleated mature human oocyteand/or fusing the diploid human somatic cell nuclear genome with theenucleated mature human oocyte to form a reconstructed oocyte, (d)activating the reconstructed oocyte using a calcium ionophore and one ormore agents that rapidly inactivate meiotic kinases and promote entryinto interphase, and (e) contacting the reconstructed oocyte with ahistone deacetylase inhibitor, thereby producing a diploid human nucleartransfer embryo.

In another embodiment the present invention provides a method forproducing a diploid human nuclear transfer embryo capable of developinginto a blastocyst containing an inner cell mass, the method comprising:(a) obtaining a diploid human somatic cell nuclear genome, (b) obtainingan enucleated mature human oocyte, (c) fusing the diploid human somaticcell nuclear genome with the enucleated mature human oocyte using afusogenic agent, to form a reconstructed oocyte, (d) subsequent to stepc, activating the reconstructed oocyte using ionomycin, (e) subsequentto step d., contacting the reconstructed oocyte with one or more agentsthat rapidly inactivate meiotic kinases and promote entry intointerphase, and one or more histone deacetylase (HDAC) inhibitors forapproximately 4 to 4.5 hours, and (f) subsequent to step e., culturingthe reconstructed oocyte in the presence of the HDAC inhibitors for anadditional approximately 10-16 hours, thereby producing a diploid humannuclear transfer embryo.

In yet another embodiment, the present invention provides a method forproducing a diploid human nuclear transfer embryo capable of developinginto a blastocyst containing an inner cell mass, the method comprising:(a) obtaining a diploid human somatic cell nuclear genome, (b) obtainingan enucleated mature human oocyte, (c) fusing the diploid human somaticcell nuclear genome with the enucleated mature human oocyte usinginactivated Sendai virus at around the lowest concentration at whichfusion still occurs, (d) approximately 1-3 hours following step c,activating the reconstructed oocyte using approximately 3 μM ionomycinfor approximately 5 minutes at approximately 37 degrees Celsius, (e)subsequent to step d., contacting the reconstructed oocyte withapproximately 10 μM puromycin, approximately 2 mM 6-DMAP, and thehistone deacetylase (HDAC) inhibitors Scriptaid and Nch51 forapproximately 4 to 4.5 hours, and (f) subsequent to step e., culturingthe reconstructed oocyte in the present of the HDAC inhibitors for anadditional approximately 10-16 hours, thereby producing a diploid humannuclear transfer embryo.

In another embodiment the present invention provides a method forproducing a diploid human nuclear transfer embryo capable of developinginto a blastocyst, the method comprising: (a) obtaining a diploid humansomatic cell nuclear genome, (b) obtaining an enucleated mature humanoocyte, (c) transferring the diploid human somatic cell nuclear genomeinto the enucleated mature human oocyte to form a reconstructed oocyte,(d) activating the reconstructed oocyte, and (e) contacting thereconstructed oocyte with a histone deacetylase inhibitor.

In another embodiment, the present invention provides a method forproducing a diploid human pluripotent stem cell from a diploid humansomatic cell, the method comprising: (a) obtaining a diploid humansomatic cell nuclear genome from a diploid human somatic cell, (b)obtaining an enucleated mature human oocyte, (c) transferring thediploid human somatic cell nuclear genome into the enucleated maturehuman oocyte to form a reconstructed oocyte, (d) activating thereconstructed oocyte using a calcium ionophore and one or more agentsthat rapidly inactivate meiotic kinases and promote entry intointerphase, (e) contacting the reconstructed oocyte with a histonedeacetylase inhibitor, thereby producing a diploid human nucleartransfer embryo, (f) culturing the diploid human nuclear transfer embryoin a medium that comprises FBS until it develops to into a blastocyst,(g) obtaining cells from the inner cell mass of the blastocyst, and (h)culturing the cells from the inner cell mass of the blastocyst to form apopulation of a diploid human pluripotent stem cells.

In another embodiment the present invention provides a method forproducing a diploid human pluripotent stem cell from a diploid humansomatic cell, the method comprising: (a) obtaining a diploid humansomatic cell nuclear genome from a diploid human somatic cell, (b)obtaining an enucleated mature human oocyte, (c) fusing the diploidhuman somatic cell nuclear genome with the enucleated mature humanoocyte using inactivated Sendai virus at around the lowest concentrationat which fusion still occurs, to form a reconstructed oocyte, (d)approximately 1-3 hours following step c, activating the reconstructedoocyte using approximately 3 μM ionomycin for approximately 5 minutes atapproximately 37 degrees Celsius, (e) contacting the reconstructedoocyte with approximately 10 μM puromycin, 2 mM 6-DMAP, and the histonedeacetylase (HDAC) inhibitors Scriptaid and Nch51 for approximately 4 to4.5 hours, (f) subsequently culturing the reconstructed oocyte in thepresent of the HDAC inhibitors for an additional approximately 10-16hours, thereby producing a diploid human nuclear transfer embryo, (g)culturing the diploid human nuclear transfer embryo in a medium thatcomprises FBS until it develops to into a blastocyst, (h) obtainingcells from the inner cell mass of the blastocyst, and (i) culturing thecells from the inner cell mass of the blastocyst to form a population ofa diploid human pluripotent stem cells.

In some embodiments the methods described herein may be modified suchthat the somatic cell nuclear genome is not transferred into an oocytethat has already been enucleated, but rather the somatic cell nucleargenome is transferred into a non-enucleated oocyte and the oocyte genomeis then removed subsequently. For example, in one embodiment the presentinvention provides a method for producing a diploid human nucleartransfer embryo capable of developing into a blastocyst containing aninner cell mass, the method comprising: (a) obtaining a diploid humansomatic cell nuclear genome, (b) obtaining a non-enucleated mature humanoocyte, (c) transferring the diploid human somatic cell nuclear genomeinto the non-enucleated mature human oocyte and/or fusing the diploidhuman somatic cell nuclear genome with the enucleated mature humanoocyte to form a triploid reconstructed oocyte, (d) subsequentlyremoving the oocyte genome from the reconstructed oocyte, (e)subsequently activating the reconstructed oocyte using a calciumionophore and one or more agents that rapidly inactivate meiotic kinasesand promote entry into interphase, and (f) subsequently contacting thereconstructed oocyte with a histone deacetylase inhibitor, therebyproducing a diploid human nuclear transfer embryo.

In some embodiments the present invention provides methods forgenerating pluripotent stem cells (e.g., ES cells) from blastocyst stageembryos generated using the methods described herein. Such stem cellscan be generated from the inner cell mass of a blastocyst stage embryomade according to the methods of the invention. Methods for generatingpluripotent stem cells (such as ES cells) from blastocyst stage embryosare known in the art and any suitable such methods can be used. Forexample, in one embodiment the inner cell mass of a blastocyst madeusing the methods of the invention is contacted with a human embryonicstem cell medium that comprises Rho kinase inhibitors, Y27632, andthiazovivin until an outgrowth of pluripotent stem cells is observed. Insome embodiments this may take about 3 to about 14 days. In someembodiments this may take about 4 days. In another embodiment thetrophectoderm of a blastocyst made using the methods of the invention isablated, for example using a laser, and cells from the inner cell massof the blastocyst are plated on a fibroblast feeder layer in humanembryonic stem cell media supplemented with thiazovivin and Rockinhibitor. Any remaining non-inner cell mass cells may be ablated with alaser at this stage also. Such methods can result in an outgrowth ofpluripotent stem cells from the plated inner cell mass, which can beexpanded to form a population of pluripotent stem cells, which may bepassaged and/or cryopreserved if desired.

In some embodiments the present invention provides human pluripotentstem cells (such as ES cells) made using the methods described herein.Such pluripotent stem cells (such as ES cells) are diploid and comprisea nuclear genome derived from a diploid human somatic cell.

In some embodiments the present invention also provides differentiatedcells generated from such pluripotent stem cells, including, but notlimited to, insulin producing cells, neurons, liver cells, heart cells,bone cells, gut cells, skin cells, hormone producing cells and bloodcells.

In one embodiment the present invention provides a reconstructed humanoocyte comprising a diploid nuclear genome obtained from a postnatalhuman somatic cell and cytoplasm from an enucleated mature human oocyte.

In one embodiment the present invention provides a diploid human nucleartransfer embryo comprising a diploid nuclear genome obtained from apostnatal human somatic cell, such as an adult human somatic cell.

In one embodiment the present invention provides a diploid humanembryonic stem cell line comprising a diploid nuclear genome obtainedfrom a postnatal human somatic cell, such as an adult human somaticcell.

In one embodiment the present invention provides a differentiated humancell derived from a human embryonic stem cell comprising a diploidnuclear genome obtained from a postnatal human somatic cell, such as anadult human somatic cell. In one such embodiment the present inventionprovides an insulin-secreting cell derived from a human embryonic stemcell comprising a diploid nuclear genome obtained from a postnatal humansomatic cell, such as an adult human somatic cell. In one suchembodiment the somatic cell is obtained from a postnatal human subjecthaving diabetes, such as an adult human subject having type I diabetes.A kit for use in a nuclear transfer method comprising: a calcium-freenuclear transfer medium.

In some embodiments the present invention provides kits comprisingcompositions and reagents useful in performing nuclear transfer methods.Such kits, and the compositions and reagents they contain, were inventedin the course of developing the improved human somatic cell nucleartransfer protocols described herein. However, such kits, and thecompositions and reagents they contain, may also be useful in othernuclear transfer applications, such as in protocols for nuclear transferusing non-human cells and in protocols for nuclear transfer usingnon-somatic cells, such as oocyte nuclear transfer protocols.

In one such embodiment the present invention provides a kit for use in anuclear transfer method, the kit comprising one or more of the followingcomponents: (a) a nuclear transfer medium (wherein the nuclear transfermedium is calcium free and/or comprises a calcium chelator, a proteinphosphatase inhibitor, and/or a fusogenic agent), (b) an activationmedium (wherein the activation medium comprises one or more of a calciumionophore, a protein translation inhibitor, and a meiotic kinaseinhibitor), (c) an embryo culture medium (wherein the embryo culturemedium comprises one or more of a histone deacetylase inhibitor, ahistone methylation inhibitor, a protein translation inhibitor, and ameiotic kinase inhibitor), and (d) an ES cell derivation medium (whereinthe ES cell derivation medium comprises fetal bovine serum (FBS)). Insome such embodiments two or more of the above components are used. Insome such embodiments three or more of the above components are used. Insome such embodiments four or more of the above components are used.

These and other embodiments of the present invention are describedthroughout the specification, claims, and drawings of the present patentapplication.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 | Efficiency of parthenogenetic development beyond the cleavagestage. Shown is the percentage of oocytes giving rise to stem celllines, blastocysts but no stem cell lines, and morulas as the percentageof the number of oocytes cleaved. Data are from references 14 and 16(see Reference List) and displayed here in a direct comparison.

FIGS. 2A-2C | Developmental potential of somatic cell nuclear transferoocytes. a, percentage of cleaved oocytes developing beyond the cleavagestage. The total number of oocytes and the number of ooycte donors (inparenthesis) contributing to a particular experiment is indicated aboveeach column. S=short treatment during the manipulation as in13,e=extended treatment, including during oocyte transport. b, expressionof a GFP transgene contained in the genome of the adult skin fibroblastused for transfer, at the cleavage stage and at the blastocyst stage. c,Cluster analysis of global gene expression profile after nucleartransfer of adult somatic cells, as well as oocytes and IVF embryos. *Data are from previous publications14 and 36 (see Reference List) andserve for comparison to the new conditions.

FIGS. 3A-3C | Development to the blastocyst stage and transcriptionalactivation of the transferred genome a, Blastocyst derived after nucleartransfer of a BJ fibroblast genome (neonatal foreskin fibroblasts). b,Blastocysts derived after nuclear transfer of an adult skin fibroblastgenome. c, ES cell outgrowth. Time post blastocyst plating is indicated.

FIGS. 4A-4H | Chromosome condensation and spindle assembly after somaticcell nuclear transfer. a, Spindle assembly on a somatic G1/G0 genome.Time indicates hours post transfer. pH3, phosphorylated histone H3. b,Spindle of the human MII oocyte. c, Somatic genome in an oocyte thatfailed to assemble a birefringent spindle around somatic chromatin. Thesomatic genome was transferred using undiluted Sendai virus. Note thelack of phosphorylated histone H3. d, Oocyte genome in the same egg.Note the segregation of oocyte chromosomes. e-h, Fluorescence of thecalcium indicator dye fluo-4 in oocyte karyoplasts before and afterincubation with fusogenic Sendai virus (less than 20 s). Time afterexposure is indicated. Arrows point to sites of fusion.

FIGS. 5A-5B | Fluorescence imaging with the calcium-responsive dyefluo-4. a, Human oocytes were incubated in medium containing fluo-4 for30 minutes, imaged for fluorescence, and concentrated Sendai virus wasadded below the plasma membrane. Shown are two oocytes for eachcondition or time point. Note that in the absence of calcium in themedium, fluorescence did not increase, while a small increase influorescence appears to occur in calcium-containing medium. Time pointafter addition of the virus is indicated. b, incubation of a humanoocyte in 3 μM of the calcium ionophore ionomycin as a positive control.

FIGS. 6A-6B | Somatic cell nuclear transfer in the absence of calcium.a, Immunochemistry to determine chromosome condensation and histonephosphorylation after transfer of a somatic cell at interphase. b, Highquality blastocysts obtained after nuclear transfer with themanipulations conducted in the absence of calcium.

FIGS. 7A-7B | Effect of FBS on blastocyst morphology and ES cellderivation. a, Blastocyst generated by somatic cell nuclear transfer inthe presence or absence of FBS. b, ICM 6 days post plating. Arrows pointto laser marks used to ablate the remaining trophectoderm cells.

FIG. 8 | Karyotypes and pluripotency marker expression in three NT-EScell lines derived from male foreskin BJ fibroblasts. The somatic donorcell used for transfer carries a GFP transgene.

FIGS. 9A-9I, 9K-9L | Derivation of diploid NT-ES cells from neonatal andadult somatic cells. a-c Characterization of an NT-ES cell line fromadult somatic cells of a female type 1 diabetic (ID 1018) a, karyotype,b,c, expression of pluripotency markers. d-f microarray analysis indermal fibroblasts, and NT-ES cell lines NT-ES5, NT-ES6, NT-ES8 andNT-ES 1018, and in ES and iPS cells. d, Expression of pluripotencymarkers. e, expression of fibroblast specific genes. f, Principlecomponent (PC) analysis of global gene expression patterns of NT-EScells, ES cells from normally fertilized embryos, iPS cells andfibroblasts. g, Teratoma analysis, h, directed differentiation intoneurons according to34. i, Directed differentiation into pancreaticprecursor cells and insulin producing cells, k, that are able to secreteinsulin into the medium upon stimulation with potassium,1, according toreference 33.

FIG. 10 | Differentiation of NT-ES cell lines made from male foreskin BJfibroblasts in embryoid bodies and in teratomas.

FIGS. 11A-11D | Retrospective analysis of the developmental potential ofnuclear transfer oocytes. Shown is the percentage of oocytes developingbeyond the cleavage stage, as percentage of eggs progressing beyond the1-cell stage. Because oocytes of a donor were used to compare twodifferent conditions, if for a particular comparison the added number ofoocyte donors exceeds 18, it indicates that these conditions were testedin parallel using oocytes of the same donor. The total number of oocytesused for analysis remained constant. a, average of the 154 oocytes of 18donors. The total number of 154 oocytes is not equal to the number ofoocytes donated by the 18 donors, but is the number of oocytes used forthe study of developmental potential after somatic cell nucleartransfer. b, Analysis with regard to factors relevant to the hormonaltreatment of oocyte donor. c, Factors relevant to the manipulation. d,Analysis regarding cell source and use of FBS for culture. n.s, nonsignificant. Statistical analysis using Chi-square test was performed bycomparing the total number of cells formed in each condition. Morulaswere assigned 15 cells, blastocysts or blastocysts that gave rise tostem cell lines 30 cells, reflecting the estimated minimal cell countfor each group.

FIGS. 12A-12T. | Chromosome segregation errors in mitosis are asignificant limitation to development after nuclear transfer. a,schematic of mitotic cell genome transfer. The oocyte genome is removedand replaced with a fibroblast in mitosis. The oocyte is allowed toassemble a spindle, is activated to complete the second meioticdivision, thereby entering the first interphase. Cell division isanalyzed at the first mitosis and in subsequent divisions. b, somaticcell genome immediately after transfer, and after 2h (c, d, e) anaphaseof somatic chromatin. f, segregation of somatic chromatin into apseudo-polar body. g, at early interphase with a single nucleus andpolar body. h, array analysis of copy number and heterozygosity. i,array analysis of copy number and heterozygosity in nuclear transferblastomere. Representative sample. j, percentage of karyotypicallyabnormal pseudo-polar bodies and blastomeres. k, Average number ofabnormalities per blastomere in NT embryos. Top label indicates the cellcycle of the transferred genome. l, quantification of multinucleation innuclear transfer blastomeres and in IVF embryos. m, somatic cell genomeat first mitotic metaphase. Arrowhead points to unattached chromosomelacking centromere. n, Bridge formation at first anaphase. o, chromosomefragments at first anaphase. p, chromosome string spanning midbody.Arrow indicates high pH3 staining on chromatin at midbody. q,phosphorylated γH2ax foci (arrowheads) indicating DNA damage at mitosis.Arrow points to the metaphase plate. r, phosphorylated γH2ax foci atinterphase. s, Multinucleation and replication protein A (RPA) foci ininterphase blastomere. t Centromere negative micronucleus in aninterphase blastomere.

FIGS. 13A-13I, 13K-13M. Segregation errors can be reduced usingepigenetic modifiers. All transfers were performed into enucleated mouseoocytes. a, percentage of nuclear transfer embryos with an error inchromosome segregation. Shown is the dependence of segregation error onthe source of origin of the nuclear genome. Note that few errors occurafter transfer of oocyte genomes, mitotic errors are slightly elevatedafter transfer of embryonic stem cell genomes, and are greatly increasedupon transfer of somatic cell genomes. b, normal chromosome segregationafter nuclear transfer of a mitotic fibroblast genome. c, chromosomebridge at the first mitosis. d-g, different types of segregationabnormalities, and the localization of the centromeres. h, chromatintrapped in the midbody in a 2-cell stage embryo. The interphase nucleusis circled. Note the persistent phosphorylation of histone H3 in thevicinity of the chromosome passenger complex component survivin. i, DNAdamage in a cleavage stage embryo. k, frequency of a particularsegregation error in affected embryos. l, segregation errors upontreatment with HDAC inhibitor and kinase inhibitor. Note that histonedeacetylation inhibitor reduces the frequency of segregation errors. m,mitotic chromosome segregation upon transfer of somatic cells afteroocyte activation.

DETAILED DESCRIPTION OF THE INVENTION

A variety of techniques for somatic cell nuclear transfer and for thegeneration of somatic cell nuclear transfer embryos were previouslyknown in the art, for example those that have been used for the cloningof animal species, including sheep (Wilmut et al., 1997 Nature 385,810-813; WO 97 07669), mice (Wakayama et al., 1998 Nature 394, 369-374;WO 99 37143), cattle (Wells et al., 1999 Biol. Reprod. 60, 996-1005),goats (Baguisi et al., 1999 Nature Biotechnol. 17, 456-461; WO 0025578), pigs (Polejaeva et al., 2000 Nature 407, 86-90) and rabbits(Chesne et al., 2002 Nature Biotechnol. 20, 366-369). Methods fornuclear transfer had also been described previously by Campbell et al.(Nuclear transfer in practice, School of Biosciences, University ofNottingham, Leicestershire, United Kingdom). However, to the best of theinventors' knowledge, prior to the present invention nobody hadsuccessfully produced a diploid human nuclear transfer embryo containinga diploid human genome from a postnatal or adult human somatic cell thatwas capable of developing into a blastocyst-stage embryo, and from whichembryonic stem (ES) cells (or lines of ES cells) could be reliablyobtained. The present invention provides various modifications andimprovements to previously used nuclear transfer techniques that allowsuch human nuclear transfer embryos and ES cell lines to be reliablyobtained—even when using adult somatic cells. For example, some of themodified and improved nuclear transfer methods described herein havebeen designed specifically to: (a) allow maintenance of plasma membraneintegrity during oocyte preparation and during cell /nuclear fusion, (b)minimize the negative consequences of compromised plasma membraneintegrity, should it occur, (c) enable rapid and efficient activation ofoocytes after nuclear transfer, and (d) maximize reprogramming byimproving replication and segregation of the somatic cell genome in theactivated egg.

The major embodiments of the present invention are described in theabove “Summary of the Invention” section of this application, as well asin the Detailed Description, Examples, Figures, and Claims sections ofthis patent application. All of such sections of this patent applicationare intended to be read together and in conjunction with one another,and the various embodiments described herein are intended to be combinedin various ways, as will be apparent to those of skill in the art.

As used herein, the term “pluripotent stem cell” refers to a cellcapable of differentiating into tissues of all three germ or dermallayers: mesoderm, endoderm, and ectoderm, unless otherwise specified.

As used herein the term “reconstructed oocyte” refers to an oocyte thathas been enucleated (i.e., had its native nuclear genome removed) andinto which a diploid somatic cell nuclear genome has been introduced.

As used herein, the term “embryo” refers to an activated oocyte that hasdivided to the two cell stage or beyond, e.g., to the four-cell,eight-cell, sixteen-cell or higher stages of embryonic development,unless otherwise specified.

As used herein, the phrase, “nuclear transfer embryo” refers to anembryo produced by inserting a nuclear genome derived from a somaticcell into an oocyte, or to an embryo produced from a “reconstructedoocyte” as defined above. The somatic cell will typically be obtainedfrom a postnatal human, such as a human child or adult. A “nucleartransfer embryo” according to the invention is distinguished from aconventionally produced human embryo, such as that produced by thepenetration of an ovum by a sperm.

Oocytes

As described elsewhere herein, the present invention provides variousmodifications and improvements to previously used somatic cell nucleartransfer techniques which allow human nuclear transfer embryos and humanES cell lines to be reliably obtained. Some of these improvementsinvolve optimizing the preparation and handling of oocytes for use inthe nuclear transfer methods of the invention, for example in order tomaintain plasma membrane integrity of the oocyte, and/or in order tominimize calcium influx through a compromised oocyte plasma membraneintegrity and/or minimize any negative consequences of such calciuminflux. Some of the main ways in which this can be achieved aredescribed below in the “nuclear transfer” section—which describes waysin which both oocytes and somatic cells can be handled in order to avoidcompromising membrane integrity, minimize calcium influx, and minimizethe negative consequences of any such calcium influx. In someembodiments, the present invention also provides other improved methodsfor handling oocytes that can result in improved oocyte and embryodevelopment. For example, in embodiments where enzymes are used duringoocyte preparation to remove cumulus cells (e.g., cumulase enzymes),exposure to such enzymes is minimized. For example, in one embodiment,after treatment with cumulase or some other suitable enzyme, the cumuluscells are mechanically removed and then the enzyme is removed.

Oocytes for use in the methods of the present invention can be obtainedfrom any suitable source. For example oocytes may be obtained from humanpatients who have given their informed consent for the use of theiroocytes in the methods described herein. Such patients may be, forexample, undergoing fertility treatments, such as in vitro fertilization(IVF) treatments and/or other assisted reproduction techniques.

Exemplary methods for obtaining and manipulating human oocytes are wellknown in the art, and any such suitable method can be used. For example,in some embodiments human oocytes are obtained after controlled ovarianstimulation (COH), such as is routinely performed in in vitrofertilization clinics. For example, oocytes may be obtained followingstimulation of ovulation with a hormone such as human chorionicgonadotropin (“hCG”). In some embodiments oocytes may be obtained from ahuman subject 30 to 40 hours after administration of an ovulationstimulus, such as hCG or leuprolide acetate, to the subject. In someembodiments the oocytes may be obtained from a human subject less than36 hours after administration of an ovulation stimulus, or 35 hoursafter administration of an ovulation stimulus, or less than 35 hoursafter administration of an ovulation stimulus. One exemplary COHprotocol consists of daily subcutaneous rFSH (recombinant folliclestimulating hormone) injections starting on day 2 of the menstrual cyclewith the addition of a daily GnRH antagonist (such as Ganirelix)starting on day 6 of stimulation. Final oocyte maturation and ovulationmay be triggered after reaching a follicle size of 18 mm by treatmentwith 4 mg of a GnRH agonist (such as Lupron) and 1000 IU of hCG (humanchorionic gonadotropin). Using such methods, oocytes may be retrieved 35hours post induction of ovulation.

In some embodiments of the present invention the oocytes used may havebeen cryopreserved prior to use (for example in the form of an intactoocyte or after enucleation). In alternative embodiments fresh (i.e.,not previously cryopreserved) oocytes or enucleated oocytes may be usedin the methods of the invention. When frozen oocytes are used anysuitable freezing (cryopreservation) method known in the art may beused. For example, cryopreservation techniques are routinely used infertility clinics to preserve and/or bank human oocytes for use in IVFprocedures, and such methods can be used in conjunction with the presentinvention.

Methods for enucleating oocytes are well known in the art. Exemplarymethods for enucleation of oocytes are provided in the Examples sectionof this patent application.

Somatic Cells

Somatic cells for use in the nuclear transfer methods described hereincan be any suitable somatic cells. In some embodiments the somatic cellsare from a postnatal human. In some embodiments the somatic cells arefrom an adult human. Any suitable type of somatic cell can be used,including, but not limited to, fibroblasts and the like. In someembodiments of the present invention, the diploid human somatic cell, orthe genome therefrom, is at the G0 or G1 stage of its cell cycle.Somatic cells in the G0 stage may be obtained by growing the cell toconfluence in vitro (typically less than 0.5% of the cells will be inS-phase after growth to confluence). In other embodiments the diploidhuman somatic cell, or the genome therefrom, is at the M (or mitosis)stage of its cell cycle. As described below in the oocyte activationsection, when M-phase somatic cells (or nuclear genomes) are used,oocytes may be activated with medium containing a translation inhibitor(such as puromycin) for a suitable time (such as 4 hours), without theneed to also use a meiotic kinase inhibitor (such as 6-DMAP). In someembodiments the entire somatic cell, including the nuclear genome andthe somatic cytoplasm, is transferred into the enucleated oocyte. Thesomatic cell nuclear genome comprises the nuclear DNA of a somatic cell,for example in the form of chromosomes, and may be within an intactsomatic cells, a nucleus, or a “karyoplast” that comprises the nuclearDNA and a small amount of cytoplasm surrounded by a membrane, such asnuclear membrane and/or cell membrane.

Methods for obtaining and culturing somatic cells are well known in theart. Exemplary methods for obtaining and culturing somatic cells areprovided in the Examples section of this patent application.

Nuclear Transfer

In some embodiments the methods of the present invention involve thetransfer of a diploid nuclear genome from a somatic cell into anenucleated oocyte. The diploid nuclear genome can be transferred in avariety of different forms. For example, and as illustrated in theExamples section of this patent application, the diploid nuclear genomecan be located within the somatic cell when it is transferred—such theentire somatic cell is introduced into, or fused with, the enucleatedoocyte. However, in some embodiments the diploid nuclear genome mayfirst be removed from the somatic cell prior to transfer. In suchembodiments the nuclear genome may comprise only the nuclear DNA (forexample in the form of chromosomes, such as metaphase chromosomes), ormay be within a nucleus, or may be within a “karyoplast” that comprisesthe nuclear DNA and a small amount of cytoplasm surrounded by amembrane. Regardless of whether the somatic cell nuclear genome used inthe methods of the present invention is present in a whole somatic cell,in a cell nucleus, in a karyoplast, or in some other form, the methodsdescribed herein may be referred to interchangeably as “nucleartransfer” methods or “cell fusion methods” and these terms are notintended to be limiting in any way.

As described elsewhere herein, an important aspect of the nucleartransfer methods of the present invention is that the protocols havebeen optimized to minimize membrane damage and to maintain the integrityof the meiotic arrest during the nuclear transfer process. Asdemonstrated in the Examples section of this patent application,compromised membrane integrity can result in unwanted calcium influx,which can in turn, compromise the meiotic state. Such membrane damageand calcium influx can be caused, for example, by the agents and methodsused to promote nuclear transfer/cell fusion.

In some of the embodiments described herein, the step of transferringand/or fusing a diploid human somatic cell, or nuclear genome therefrom(e.g., in a nucleus or in a karyoplast), into/with an enucleated maturehuman oocyte comprises using a fusogenic agent. The term “fusogenicagent” is used herein to refer collectively to fusion-promotingchemicals and other agents (such as inactivated viruses, portions ofinactivated viruses, or proteins derived from viruses) that can be usedto fuse a somatic cell (or nucleus, nuclear genome, or karyoplast from asomatic cell) with an oocyte. In embodiments where a fusogenic agent isused, any suitable fusogenic agent known in the art may be used. In oneembodiment the fusogenic agent may be polyethylene glycol. In oneembodiment the fusogenic agent may be a Sendai virus, such as aninactivated Sendai virus. In some embodiments inactivated Sendai virusHVJ-E may be used. As described above, it is a particular finding of thepresent invention that exposure to fusogenic agents can be controlled soas to minimize membrane damage and the associated unwanted influx ofcalcium during and after the nuclear transfer process. Thus, in someembodiments the fusogenic agent, such as inactivated Sendai virus, isused at a low concentration, and preferably at the minimum dosage,amount, or concentration that can be readily used while still allowingfusion to occur. Using the fusogenic agent, such as Sendai virus, at alow concentration helps to minimize exposure to the fusogenic agent andis desirable because fusogenic agents can compromise membrane integrityleading to undesirable calcium influx. Using a low concentration of thefusogenic agent, such as Sendai virus, minimizes this source of calciuminflux into the oocyte during the nuclear transfer process. Thus in someembodiments the fusogenic agent used, and/or the amount of the fusogenicagent used, is selected so as to minimize or eliminate calcium influxinto the oocyte during the nuclear transfer process. One of skill in theart can readily determine a suitable amount of a fusogenic agent to useby, for example, performing a dose/response study and looking at theeffects of the fusogenic agent on calcium influx and/or fusion. In oneembodiment of the invention, where inactivated Sendai virus is used asthe fusogenic agent, Sendai virus HVE-J from Genome One Cosmobio isreconstituted according to the manufacturer's instructions and thendiluted 1:10 to 1:20 in a suitable medium. The resulting concentrationof the inactivated Sendai virus HVE-J is low but sufficient to inducefusion. Similarly, in some embodiments exposure to the fusogenic agentis minimized in other ways, for example by only exposing a small area ofthe somatic cell or oocyte to the fusogenic agent, for example onlyexposing one side of the cell or one portion of the cell to thefusogenic agent. In one such embodiment, exposure to the fusogenic agentmay be minimized by first aspirating the somatic cell into the pipetteto be used for nuclear transfer/cell fusion and then subsequentlyaspirating fusogenic agent (such as Sendai virus) into the pipette, suchthat the somatic cell will then only be exposed to the fusogenic agenton one side. The side of the somatic cell exposed to the fusogenic agentmay then be juxtaposed with the plasma membrane of the oocyte to allownuclear transfer/cell fusion. In this way, injection of fusogenic agentbelow the plasma membrane of the somatic cell and/or the oocyte isavoided.

In some embodiments of the present invention nuclear transfer/cellfusion may be achieved using an “electro-fusion” method that comprisesadministering an electrical pulse. However, in other embodiments thepresent methods do not comprise using electrofusion or administering anelectrical pulse during the nuclear transfer/cell fusion step.

Whichever method is used to perform the nuclear transfer/cell fusionstep (e.g., whether a fusogenic agent is used, electro-fusion isperformed, or some other method is used), the present invention providesmethods and compositions that can be used during and after the nucleartransfer/cell fusion step to minimize the detrimental effects of calciuminflux that can otherwise occur during these procedures. Thus, in someof the embodiments of the present invention the step of transferringand/or fusing a diploid human somatic cell genome into/with theenucleated mature human oocyte is performed in a calcium-free medium,which may be referred to as a calcium-free nuclear transfer medium or acalcium-free cell fusion medium. Such a calcium-free medium may be usedduring and after nuclear transfer/cell fusion, and may be used until theoocyte is activated, or up to about one hour or two hours prior toactivation. For example, and as shown in the Examples herein, it hasbeen found that use of a calcium-free medium during nuclear transferresults in significantly improved results, including improveddevelopmental potential of the resulting reconstructed oocytes andnuclear transfer embryos, improved frequency of derivation of diploidstem cell lines (ES cell lines), and improved ability to derive ES cellslines from both postnatal and adult human somatic cells—which had notbeen achieved previously by others using other methods. Exemplarycalcium-free nuclear transfer media are described in the Examplesherein. In addition, one of skill in the art can readily preparecalcium-free media using principles known in the art. In someembodiments, in addition to, or instead of, using a calcium-free nucleartransfer medium, a calcium chelator may be used during and after nucleartransfer/cell fusion, and may be used until the oocyte is activated, orup to about one hour or two hours prior to activation. Suitable calciumchelators include, but are not limited to, ethylene diamine tetra-aceticacid (EDTA), ethylene glycol tetra-acetic acid (EGTA),1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), andother calcium chelators known in the art. In some embodiments, acell-permeant calcium chelator may be used, such as BAPTA-AM. Forexample, in one exemplary but non-limiting embodiment, BAPTA-AM may beused at a concentration of about 1-5 μM for up 20 minutes prior tonuclear transfer/cell fusion. The BAPTA-AM acts as an intracellularcalcium chelator, thereby clamping the intracellular calciumconcentrations during the nuclear transfer/cell fusion process. In someembodiments, in addition to, or instead of, using a calcium-free mediumand/or a calcium chelator, one or more inhibitors of proteinphosphatases can be used both during and after nuclear transfer/cellfusion, and may be used until the oocyte is activated, or up to aboutone hour or two hours prior to activation. Such protein phosphatases actdownstream of calcium influx and can help to mitigate the effects of anycalcium influx during the nuclear transfer/cell fusion process. Anysuitable protein phosphatase known in the art may be used, including,but not limited to, okadaic acid.

Other exemplary nuclear transfer/cell fusion protocols are provided inthe Summary of the Invention, Examples, and Claims of this application,and/or are known in the art, and may be used in conjunction with thepresent invention.

Activation of Reconstructed Oocytes and Subsequent Embryo Culture

As described elsewhere herein, the present invention provides variousmodifications and improvements to previously used somatic cell nucleartransfer techniques which allow human nuclear transfer embryos and humanES cell lines to be reliably obtained. Some of these improvementsinvolve the step in which reconstructed oocytes are activated—aftercompletion of the nuclear transfer /cell fusion step. In particular, insome embodiments the present invention provides improved methods thatallow for the rapid and efficient activation of reconstructed oocytesand which lead to improved efficiencies of embryo generation and of EScell line generation.

In some embodiments of the present invention the step of activating thereconstructed oocytes comprises delivering a calcium pulse to thereconstructed oocyte, for example using a calcium ionophore. Anysuitable calcium ionophore may be used, including, but not limited to ofA23187 and ionomycin. The calcium pulse may be repeated up to 10 timesover the time course of 3 hours, or until the oocyte enters interphase.In such embodiments the reconstructed oocyte must be placed in acalcium-containing medium. Therefore, in embodiments where acalcium-free medium was used during the prior nuclear transfer/cellfusion step, the medium must be changed to a calcium-containing mediumprior to, or concurrently with, contacting the oocyte with the calciumionophore. In some such embodiments, the medium is changed to acalcium-containing medium about 15 minutes or about 30 minutes prior tocontact with the calcium ionophore.

In some embodiments of the present invention the step of activating thereconstructed oocytes comprises contacting the oocyte with a translationinhibitor, or a meiotic kinase inhibitor, or, in some embodiments, botha translation inhibitor and a meiotic kinase inhibitor. Such methodspromote entry into interphase. In some embodiments the translationinhibitor and/or meiotic kinase inhibitor may be used together with acalcium ionophore treatment, and may be used concurrently with theionophore treatment and also after the ionophore treatment. Translationinhibitors and meiotic kinase inhibitors are known in the art and anysuitable such agents can be used. Suitable translation inhibitorsinclude, but are not limited to, puromycin. Suitable meiotic kinaseinhibitors include, but are not limited to, 6-DMAP (the chemical namesof “6-DMAP” include 6-(dimethylamino)purine and N6,N6-dimethyladenine,and 6-DMAP also has CAS registry number 938-55-6). Any concentration oramount of these agents that is sufficient to rapidly inhibittranslation, rapidly inactivate meiotic kinases, and/or rapidly resultin oocyte activation and entry into interphase may be used. For example,in one embodiment the translation inhibitor puromycin is used atapproximately 10 μM and the meiotic kinase inhibitor 6-DMAP is used atapproximately 2 mM. However, one of skill in the art can readilydetermine suitable concentrations or amounts of these or other agents touse using standard methods known in the art, such as dose-responsestudies and the like. Similarly, one of skill in the art can determinean appropriate duration for the exposure of the oocyte to suchtranslation inhibitors and/or meiotic kinase inhibitors. In someembodiments of the present invention, the reconstructed oocyte iscontacted with the translation inhibitor and/or the meiotic kinaseinhibitor for approximately 4 to 4.5 hours.

In embodiments where the diploid human somatic cell genome is obtainedby removing the nucleus or nuclear genome from a human somatic cellduring the mitotic (M) stage of the cell cycle, a meiotic kinaseinhibitor (such as 6-DMAP) need not be used. For example, the presentinventors have found that under these circumstances, a reconstructedoocyte can be rapidly and efficiently activated by contacting it withmedium containing a translation inhibitor (such as puromycin) for 4hours without the addition of any meiotic kinase inhibitor (such as6-DMAP)—as demonstrated by efficient polar body extrusion. However, inembodiments where the diploid human somatic cell nuclear genome isobtained by removing the nucleus or nuclear genome from a human somaticcell at some other stage of its cell cycle (such as during the G1 or G0stages), it is preferred that both a translation inhibitor (such aspuromycin) and a meiotic kinase inhibitor (such as 6-DMAP) be used.

Other exemplary activation protocols are provided in the Summary of theInvention, Examples, and Claims of this application. In addition, othermethods that are known in the art to be useful for oocyte activation canbe employed in conjunction with the methods described herein. Forexample, it is known in the art that oocyte activation can be achievedby applying an electric pulse to an oocyte, by chemically induced shock,by penetration of the oocyte by sperm, or by any combination of suchmethods. In some embodiments of the present invention the step ofactivating the oocyte may comprise one of such methods, such as, forexample, administering an electrical pulse instead of, or in additionto, using a calcium ionophore. However, in other embodiments the presentmethods do not comprise using such other methods known in the art, suchas using an electrical pulse.

Histone Deacetylase Inhibitors

As described elsewhere herein, one of the improvements over and aboveprior nuclear transfer techniques that is provided by the presentinvention is that the present methods result in improved reprogrammingas a result of improved replication and segregation of the somatic cellgenome in the activated oocyte. Chromosome mis-segregation is frequentafter somatic cell nuclear transfer using other prior art methods.However, it is a discovery of the present invention that certain agents,when applied during the first cell cycle in the reconstructed oocyte,can increase the fidelity of chromosome duplication and enable efficientdevelopment of embryos to the blastocyst stage. Such agents includehistone deacetylase inhibitors and histone methylation inhibitors.

Thus, in some embodiments the methods of the present invention involvecontacting a reconstructed oocyte using one or more histone deacetylaseinhibitors. Many histone deacetylase inhibitors are known in the art andany suitable histone deaceytlase inhibitor may be used in the methods ofthe invention. In some embodiments the histone deacetylase inhibitor isscriptaid (chemical name:6-(1,3-Dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-hexanoic acidhydroxyamide, CAS No-287383-59-9). In other embodiments the histonedeacetylase inhibitor is Nch51 (chemical name:S-7-oxo-7-(4-phenylthiazol-2-ylamino)heptyl 2-methylpropanethioate, CASNo-848354-66-5). Other histone deacetylase inhibitors that may besuitable include, but are not limited to: m-carboxycinnamic acidbishydroxamide (CBHA), trichostatin A (TSA), trichostatin C,salicylihydroxamic acid (SBHA), azelaic bishydroxamic acid (ABHA),azelaic-1-hydroxamate-9-anilide (AAHA), 6-(3-chlorophenylureido) carpoichydroxamic acid (3C1-UCHA), oxamflatin, A-161906, PXD-101, LAQ-824,CHAP, MW2796, MW2996, of SAHA, CI-994, PXD-101, LBH589, FK228,MGCD-0103, R306465, PCI-24781, SB-939, ITF-2357, and MS-275. In someembodiments the histone deacetylase inhibitor is selected from the groupconsisting of Scriptaid, Nch51 and trichostatin. In some embodimentsboth Scriptaid and Nch51 are used.

In some embodiments the methods of the present invention involvecontacting a reconstructed oocyte using one or more histone methylationinhibitors. Many histone methylation inhibitors are known in the art andany suitable histone methylation inhibitor may be used in the methods ofthe invention. In one embodiment the histone methylation inhibitor isEPZ-6438. In one embodiment the histone methylation inhibitor isselected from the group consisting of EZH inhibitors, includingdeazaneplanocin A (DZNep), EPZ005687, or other compounds inhibitingPRC2, or the histone methyl transferase inhibitor BIX01294.

In some embodiments of the present invention a reconstructed oocyte iscontacted with a histone deacetylase inhibitor and/or histonemethylation inhibitor starting from the time of the first cell cycle inthe reconstructed oocyte. In some embodiments, this may includecontacting the reconstructed oocyte with the histone deacetylaseinhibitor and/or histone methylation inhibitor during the activationstep, or very soon after the activation step, or both during and afterthe activation step. In some embodiments the reconstructed oocyte, orembryo derived from the reconstructed oocyte, is contacted with thehistone deacetylase inhibitor and/or histone methylation inhibitor forat least about 14 hours post activation, or for approximately 14 toapproximately 21 hours post activation, or until just prior to the firstmitosis. In some embodiments the reconstructed oocyte, or embryo derivedfrom the reconstructed oocyte, is maintained in a medium that contains ahistone deacetylase inhibitor and/or histone methylation inhibitor forat least 8 hours post activation. In some embodiments, the reconstructedoocyte, or embryo derived from the reconstructed oocyte, is maintainedin a medium that contains a histone deacetylase inhibitor and/or histonemethylation inhibitor for about 15 to about 20 hours post activation, oruntil before the first mitosis.

In some embodiments of the present invention, subsequent to the step ofactivating the reconstructed oocyte, the reconstructed oocyte iscontacted with one or more histone deacetylase (HDAC) inhibitors and/orhistone methylation inhibitors for approximately 4 to 4.5 hours, and isthen subsequently cultured in the presence of one or more HDACinhibitors and/or histone methylation inhibitors for an additionalapproximately 10-16 hours. In some embodiments, subsequent to the stepof activating the reconstructed oocyte, the reconstructed oocyte iscontacted with the histone deacetylase (HDAC) inhibitors Scriptaid andNch51 for approximately 4 to 4.5 hours, and is then subsequentlycultured in the present of the same HDAC inhibitors for an additionalapproximately 10-16 hours. In some embodiments the reconstructed oocyteis first activated (e.g., by contacting the oocyte with a calciumionophore), and is then contacted with a medium comprising puromycin anda histodeacetylase inhibitor for about 4 hours, and is then contactedwith a medium containing a histodeacetylase inhibitor but not puromycinfor a further 11-17 (e.g., 11-13 or 15-17) hours. In some suchembodiments the medium further comprises 6-DMAP.

Caffeine

In some embodiments the methods of the present invention comprisecontacting the diploid human somatic cell genome, the enucleated humanoocyte, the reconstructed oocyte, or the diploid human nuclear transferembryo with caffeine.

In some embodiments the methods of the present invention do not comprisecontacting the diploid human somatic cell genome, the enucleated humanoocyte, the reconstructed oocyte, or the diploid human nuclear transferembryo with caffeine. Rather, and as described in other sections of thispatent application, the protocol is optimized at the level of takingsteps to maintain membrane integrity and to limit the ability of calciumto compromise the meiotic arrest of the oocyte.

Micromanipulation and Culture of Oocytes, Somatic Cells & Embryos

Instruments for micromanipulating oocytes, somatic cells, karyoplasts,embryos and the like are well known in the art. Micropipettes andneedles suitable for us in such manipulations include, but are notlimited to, those available Origio, Humagen, Cook Medica, and EppendorfMicropipettes can also be laboratory-made using a needle puller and amicroforge. Any suitable micromanipulators for manipulatingmicropipettes can be used, such as those available from Narishige,Sutter Instruments, Eppendorf and other manufacturers. Manipulations canbe performed using a microscope, such as an inverted microscope having aheated stage and equipped with any required micromanipulators. Suitablemicroscopes include, but are not limited to, the NikonTE2000-U equippedwith a 40× objective and Hoffman contrast optics, and the Olympus IX71with relief contrast optics. Other exemplary methods formicromanipulating oocytes, somatic cells, karyoplasts, embryos and thelike are provided in the Examples of the present application and/or areknown in the art.

In addition to some of the specific new methods described herein,several general methods suitable for handling of oocytes, somatic cells,karyoplasts, embryos and the like are well known in the art and can beused in conjunction with the specific methods of the present invention.For example, oocytes, somatic cells, and embryos can be handled andcultured or manipulated in physiologically suitable media known in theart. Generally oocytes, somatic cells, karyoplasts, and embryos will bemaintained at 37° C. as far as is possible. For example, after oocyteretrieval (e.g., from a patient in an IVF clinic) and/or retrieval ofsomatic cells, such cells may be transported to the site of manipulationin a portable incubator at 37° C. Suitable media for culturing somaticcells are known in the art. Suitable media for culturing andmanipulating oocytes are also known in the art. One such medium that maybe used is named “GMOPSplus” media, which is available from Vitrolife.Another suitable medium for embryo culture is Global media availablefrom IVFOnline, LLC. In general, all manipulations should be performedin media that maintain a physiological environment at ambientatmosphere, while all culture should be performed in media that maintaina physiological environment in the atmosphere of an incubator—i.e.,generally at 5% CO2. Media can be supplemented with a source of protein,e.g., human serum albumin or plasma without active complement factors(plasmanate). For example, Plasmanate, available from Talecris, may beadded to Global media at 10% volume percentage. Other media that may beemployed for manipulating oocytes includes, but is not limited to: HTF(IVFOnline, LLC or other supplier), Ham's F-10 or a modified version ofit (Irvine Scientific), Gamete Buffer (Cook medical), or other mediathat maintain physiological conditions at ambient atmosphere.Maintenance and culture of oocytes, reconstructed oocytes and nucleartransfer embryos can also be performed using other commerciallyavailable media such as ART media (LifeGlobal or IVFOnline, LLC). Suchmedia may be either single-step media (that can be used from day 1 today 7), such as Global media, or the Single Step Medium from IrvineScientific), or two-step media (that require a change on day 3 afteractivation). Examples of suitable two-step media systems include usingCook cleavage medium (from Cook Medical, Inc.) for day 1 to day 3followed by Cook Blastocyst medium (from Cook Medical, Inc.) until day 7post activation. Other two-step media systems include, but are notlimited to, P-1 medium used with the MultiBlast Medium (from IrvineScientific), and Quinn's Advantage Cleavage media used with Quinn'sAdvantage Blastocyst media (Cooper Surgical). Embryos can be culturedusing any suitable means known in the art. For example, they may becultured in small drops (e.g., around 30-50 microliters) of media, whichmay be covered with oil. Suitable oils that can be used include “oil forembryo culture” from Irvine Scientific, “culture oil” from Cook Medical,and “LiteOil” from IVFOnline. Embryos may also be cultured in smalldishes or wells, such as 4-well cell culture plates (e.g., from ThermoScientific) containing around 500 to 700 microliters of medium. Whenthis method is used there may be no need to cover the cultures with oilbecause of the larger liquid volume.

Other exemplary methods, reagents and media for handling and culture ofoocytes, somatic cells, and embryos are provided elsewhere in thisDetailed Description, and/or in the Summary of the Invention, Examples,and Claims sections of this patent application. For example, in someembodiments activated oocytes / embryos are cultured in Global totalmedium containing 10% FBS (quality controlled for compatibility withhuman ES cell growth) and an HDAC inhibitor for 12 hours, followed byculture to the blastocysts stage in medium containing 10% FBS.

Derivation of Pluripotent Stem Cells

Methods for obtaining pluripotent embryonic stem cells (ES cells) fromblastocyst stage embryos are known in the art, and any such suitablemethod may be used in accordance with the present invention. Forexample, in one embodiment the nuclear transfer embryos of the presentinvention are cultured until an inner cell mass of an embryo may beisolated after approximately six to seven days of development, or untilthe embryo has reached the expanded blastocyst stage. Pluripotent stemcells can be generated from such an inner cell mass, for example usingmethods known in the art. Analysis of gene expression and developmentalpotential can also be performed to demonstrate the pluripotency of thecells obtained, and karyotype and short tandem repeat analysis can beperformed to confirm the presence of the somatic cell genome in the stemcells. In some embodiments the inner cell mass of a blastocyst may beisolated using a laser. In some embodiments it may also be possible toobtain pluripotent stem cells without isolation of the inner cell mass,for example by plating an intact blastocyst in a dish. In someembodiments the inner cell mass of a blastocyst may be plated on a layerof suitable feeder cells, including, but not limited to, a feeder layerof mouse embryonic fibroblast cells. The feeder layer may also becomposed of human cells, or any other suitable substrate that cansupport the growth of human pluripotent stem cells. Such substratesinclude, but are not limited to, Matrigel, UV/ozone treated plasticware,gelatin-coated plastic, and the like. Any culture medium suitable forculture of pluripotent stem cells may be used, and several such mediaare known in the art. For example, the culture medium may be composed ofKnockout DMEM, 20% Knockout Serum Replacement, nonessential amino acids,2.5% FBS, Glutamax, beta-mercaptoethanol, 10 ng/microliter bFGF, andantibiotic. The employed medium may also be a variation of this medium,for example without the 2.5% FBS, or with a higher or lower % ofknockout serum replacement, or without antibiotic. The employed mediummay also be any other suitable medium that supports the growth of humanpluripotent stem cells in undifferentiated conditions, such as mTeSR(available from STEMCELL Technologies), or Nutristem (available fromStemgent), or ES medium, or any other suitable medium known in the art.Other exemplary methods for generating/obtaining pluripotent stem cellsfrom a blastocyst, such as a blastocyst made according to the methods ofthe present invention, are provided in the Summary of the Invention,Examples, and Claims of this application, and/or are known in the art.

Kits

In some embodiments the present invention provides kits comprisingcomponents and compositions useful in performing nuclear transfermethods. Such kits, and the components and compositions they contain,were invented in the course of developing the improved human somaticcell nuclear transfer protocols described herein. However, such kits,and the components and compositions they contain, may also be useful inother nuclear transfer applications, such as in protocols for nucleartransfer using non-human cells and in protocols for nuclear transferusing non-somatic cells, such as oocyte nuclear transfer protocols. Kitsaccording to the present invention may comprise any of the components orcompositions described herein in any desired combination.

For example, in one embodiment the present invention provides a kit foruse in a nuclear transfer method, the kit comprising one or more (or twoor more, or three or more, or four or more) of the following components:(a) a nuclear transfer/cell fusion component (wherein the nucleartransfer/cell fusion component comprises a calcium free medium, acalcium chelator, a protein phosphtase inhibitor, and/or a fusogenicagent), (b) an activation component (wherein the activation componentcomprises one or more of a calcium ionophore, a protein translationinhibitor, and a meiotic kinase inhibitor), (c) an embryo culturecomponent (wherein the embryo culture component comprises one or more ofa histone deacetylase inhibitor, a histone methylation inhibitor, aprotein translation inhibitor, and a meiotic kinase inhibitor), and (d)an ES cell derivation component (wherein the ES cell derivationcomponent comprises fetal bovine serum (FBS)).

In some such embodiments one or more of the “components” may be amedium, such as a nuclear transfer/cell fusion medium, an activationmedium, an embryo culture medium, and/or an ES cell derivation medium.

The invention is further described by way of the following non-limitingexamples.

EXAMPLES

Certain publications are referred to throughout the Examples section ofthis patent application. In some sections of the Examples thepublications are referred to by reference to the first author's name andthe publication year (shown in parentheses). In other sections of theExamples the publications are referred to using numerals (shown insuperscript) which refer to numbered publications. In both cases thefull citations for each of publications referred to are provided in theReference List located at the end of the Examples.

Example 1

Human pluripotent stem cells have the ability to self-renewindefinitely, and give rise to cell and tissue types of all three germlayers, enabling the study of human disease in cellular models (forreview see (Robinton and Daley, 2012)). More recently, human embryonicstem cells have been approved for use in clinical trials aimed atdetermining the safety of transplanted cells in spinal cord injury andmacular degeneration (Schwartz et al., 2012). The derivation ofpluripotent stem cells from somatic cells holds the promise to useautologous cells for cell replacement for degenerative diseases such asdiabetes or Parkinson's Disease (Tabar et al., 2008).

The transfer of somatic cell nuclei into oocytes can give rise topluripotent stem cells that are consistently equivalent to embryonicstem cells1-3, holding promise for autologous cell replacementtherapy4,5. Though methods to induce pluripotent stem cells from somaticcells by transcription factors6 are widely used in basic research,numerous differences to embryonic stem cells have been reported7-11,potentially affecting their clinical use. Because of the therapeuticpotential of diploid embryonic stem cell lines developed from adultcells of diseased human subjects, we have systematically investigatedthe parameters affecting efficiency and developmental potential in theirderivation.

Results

Nuclear transfer using an efficient activation protocol and HDACinhibitors during the first cell cycle allow development to theblastocyst stage.

We have previously reported the derivation of triploid pluripotent stemcells containing a reprogrammed somatic cell nucleus, and a haploidoocyte genome¹⁴. Development to the blastocyst stage only occurred inthe presence of the oocyte genome. Diploid nuclear transfer cellsarrested development at the cleavage stages, failing to expressembryonic genes¹⁴. To improve developmental potential of diploid nucleartransfer oocytes, we tested the effect of histone deacetylationinhibitors as well as changes to the artificial activation protocol ondevelopmental potential after nuclear transfer of neonatal and adultsomatic cells. These modifications were based on the report thatdeacetylase inhibitors improve reprogramming efficiency and developmentafter somatic cell nuclear transfer into mouse oocytes¹⁵, and on ourprevious observation that parthenogenesis was more efficient whenoocytes were activated with the translation inhibitor puromycin¹⁶ thanwhen activated with the kinase inhibitor 6-dimethylaminopurine(6-dmap)¹⁴ (FIG. 1). We first tested the use of puromycin for oocyteactivation for somatic cell nuclear transfer without removing the ooyctegenome, resulting in efficient development to the blastocyst stage (FIG.2A). However, when this activation protocol was applied to enucleatedoocytes, development still arrested at cleavage stages (FIG. 1A). Onlywhen we applied the histone deacetylation inhibitor (HDAC) scriptaid forthe first 17 hours of embryo culture after the calcium pulse, did weobserve development to the morula and blastocyst stage at a lowfrequency (FIG. 2A). An additional improvement in developmentalpotential was observed when both puromycin and 6-DMAP were combinedduring oocyte activation, resulting in development to expandedblastocysts (FIG. 2A, 2B, FIG. 3A, 3B). Puromycin promotes ooycteactivation by inhibition of translation of cyclinB^(17,18), while 6-DMAPinhibits the activity of meiotic kinases; their combined use may resultin a more complete or more rapid inactivation of meiotic kinases. Theseresults show that an improved activation protocol using both puromycinand 6-DMAP for kinase inhibition and the use of scriptaid alloweddevelopment to the blastocyst stage after somatic cell nuclear transfer.

Reprogramming of Gene Expression after Somatic Cell Nuclear Transfer

Because development beyond the cleavage stage requires geneexpression¹⁹, development of nuclear transfer oocytes to the morula andblastocyst stage indicates transcriptional activity of the transferredsomatic cell genome. The use of somatic cells containing a GFP transgeneallowed us to conveniently visualize the activity of the somatic cellgenome. While nuclear transfer protocols that did not result indevelopment beyond the cleavage stage showed no expression of the GFPtransgene¹⁴, 58% (14 of 24) of the nuclear transfer cells treated withhistone deacetylation inhibitor showed GFP expression (FIG. 2B), and hada global gene expression profile similar to IVF embryos (FIG. 2C),demonstrating that transcriptional reprogramming had occurred. From the7 blastocysts obtained using optimized nuclear transfer protocols, weattempted to derive nuclear transfer ES cells. 3 of the blastocystsformed an outgrowth (FIG. 3C), but none gave rise to an ES cell line.

More recently, it has become possible to derive diploid pluripotent stemcell lines from fetal fibroblasts¹³. The derivation of two cell linesfrom neonatal fibroblasts was also mentioned, although no karyotype orevidence of pluripotency was provided. Thus far, there is no report onthe derivation of nuclear transfer ES cell lines from any postnatalcell. While the use of the HDAC inhibitor TSA is consistent with ourapproach, the authors also attributed successful derivation to the useof caffeine during oocyte enucleation to promote nuclear envelopebreakdown and chromosome condensation, the use of a hormone stimulationprotocol yielding a small number of high quality oocytes, and to the useof an electrical pulse for oocyte activation. Because these conclusionsare based on a limited number of donors and on the use of fetalfibroblasts¹³, we determined the relevance of these modifications on thederivation of pluripotent embryonic stem cells after nuclear transfer ofpostnatal and adult somatic cells.

Maintaining Plasma Membrane Integrity

We first determined whether oocyte enucleation interfered with theability of the oocyte to condense somatic chromatin, a processcorrelating with developmental potential after somatic cell nucleartransfer²⁰. When we transferred somatic cell genomes at G1 or G0 stagesof the cell cycle into enucleated oocytes, 17 of 23 (74%) assembled aspindle within 1-4 hours after transfer as determined by microtubulebirefringence²¹ or immunostaining (FIG. 4A). Somatic chromosomes werecondensed and phosphorylated on serine28 of histone H3, but not alignedon a metaphase plate, because bipolar amphitelic attachments cannotoccur on unreplicated chromosomes, as they do in a spindle of the MIIoocyte (FIG. 4B). Though chromosome condensation did not occur in alloocytes that were enucleated and transferred with a somatic cell genome,failure to condense somatic chromatin was not related to the enucleationprocedure. Chromosome condensation and spindle assembly occurred withsimilar efficiency in oocytes only transferred with a somatic cellgenome and not enucleated ( 10/13, 77%). Two non-enucleated oocytes thathad failed to assemble a spindle around somatic chromatin were analyzedusing immunochemistry. Both oocytes showed no phosphorylation of somatichistones (FIG. 4C), and showed segregation of the oocyte genome with theformation of a midbody positive for borealin (FIG. 4D), a component ofthe chromosome passenger complex localizing to the midbody atanaphase²². In contrast to a previous report using a small number of 3enucleated oocytes¹³, an effect of oocyte enucleation on meiotic arrestwas not apparent.

Because a rise in intracellular calcium concentration is a potentinducer of anaphase in human oocytes, we investigated the effect ofSendai virus on intracellular calcium levels. During enucleation of theoocyte, the genome was removed as a karyoplast, surrounded by a smallamount of cytoplasm and plasma membrane. Intact oocytes and karyoplastswere equilibrated with the calcium indicator dye fluo-4 and exposed toinactivated Sendai virus. Within minutes, an increase of fluo-4fluorescence was observed (FIG. 4E-4H, FIG. 5). Fluorescence was calciumdependent, as oocytes incubated in calcium-free media showed decreasedfluo-4 fluorescence compared to oocytes incubated in calcium-rich media(FIG. 5). These results show that the fusion of somatic cells usingSendai virus can increase calcium influx and compromise the integrity ofmeiotic arrest. For these reasons, nuclear transfer experiments from allbut 4 oocyte donations were performed using Sendai virus diluted up to20 fold. And for two donations, calcium was omitted from the medium usedfor transfer, as well as during incubation prior to oocyte activation.This manipulation in the absence of calcium was compatible withcondensation of transferred somatic chromatin (FIG. 6A).

Additional technical adaptations, including an electrical pulse foroocyte activation and the use of caffeine during oocyte manipulationallowed development to the blastocyst stage, consistent with a previousreport¹³, though the efficiency remained at a modest 10% blastocystdevelopment. The most efficient and high quality blastocyts wereobtained when caffeine was also added during oocyte transport, combinedwith omitting calcium from the manipulation medium (FIG. 2A), withblastocysts of high quality with a distinct inner cell mass andexpanding trophectoderm (FIG. 6B).

Derivation of Diploid Nuclear Transfer ES cells from Adult and NeonatalSomatic Cells

For the derivation of nuclear transfer ES (NT-ES) cell lines, we alsoadded fetal bovine serum (FBS) to the embryo culture and derivationmedia¹³. FBS promoted the formation of an inner cell mass (ICM) at theexpense of trophectoderm cells. Of 8 nuclear transfer blastocystsgenerated without the addition of FBS, 2 lacked an ICM, and containedexclusively (60 or more) trophectoderm cells (FIG. 3). In the presenceof FBS, even embryos with a small number of cells formed a distinctinner cell mass (4 of 4), with fewer than 20 trophectoderm cells (FIG.7A). Three of four such blastocysts formed an outgrowth (FIG. 10B) anddeveloped into three cell lines containing a diploid male karyotypederived from foreskin fibroblasts (FIG. 8). An NT-ES cell line with adiploid female karyotype was derived from an adult somatic cell of atype 1 diabetic (age 32 years), (FIG. 9A). All four cell lines expressedmarkers of pluripotency (FIG. 9B-9D, FIG. 8), and lacked markers of thedermal fibroblasts (FIG. 9E). In an analysis of global gene expressionpatterns, NT-ES cells clustered closely with other human pluripotentstem cells, including ES cells from fertilized embryos and inducedpluripotent stem (iPS) cells (FIG. 9F). In embryoid bodies and upontransplantation into immuno-compromised mice, all four NT-ES cell linesgave rise to cell types of three germ layers (FIG. 9G, FIG. 10). Whenexposing them to a combination of patterning factors (Methods) we foundefficient differentiation into neurons (FIG. 9H), pancreatic andduodenal homeobox-1 (Pdx-1) positive pancreatic cells (FIG. 9I), andinto insulin positive cells (FIG. 9K) that were able to secrete insulininto the medium upon potassium stimulation (FIG. 9L).

Discussion

Though nuclear transfer blastocysts could be obtained with an efficiencyof approx. 10%, developmental efficiency varied between different oocytedonors, even when other aspects of the nuclear transfer protocol werekept constant. In an experiment of 3 oocyte donations occurring in shortsuccession within 4 days, we used identical protocols, reagents, andreplicates of the same somatic cell cultures. Oocytes of one donoryielded blastocysts with an efficiency of 44% ( 4/9 oocytes), anotheryielded cleavage stage cells only, and oocytes of a third donor resultedin a single blastocyst ( 1/12 oocytes, 8%). To better understand thesource of this variation we performed a retrospective analysis ofvariations in the hormone stimulation protocol and in nuclear transferprocedures (FIG. 13) examining 154 oocytes obtained from 18 donors(oocytes in the last four columns of FIG. 2A). In contrast to a previousreport¹³, we found no effect of the number of MII oocytes retrieved perdonation and developmental competence (FIG. 11B). In fact, thederivation of a NT-ES cell line from adult somatic cells resulted using9 oocytes for nuclear transfer of a cycle with a total of 31MII oocytes.We also observed no significant effect of the daily dose ofgonadotropin. And both gonadotropin releasing hormone (GnRH) antagonistand GnRH agonist protocols resulted in development to the blastocyststage with a similar efficiency. However, a trend towards a negativeeffect of the total duration (days) of gonadotropin stimulation requiredto reach a follicular size of 18 mm, was observed. In addition, greaterdevelopmental potential in the age group of 21-26 years in comparison toage 27-32 years was noted. Though short stimulation cycles are preferredduring in vitro fertilization (IVF) treatments²³, no significant effecton blastulation have been demonstrated. And although an effect ofmaternal age on pregnancy rates is well documented, a decrease inblastulation was shown above the age of 40²⁴. The requirements forsomatic cell reprogramming using the currently available nucleartransfer protocols may be more stringent, and reveal biologicaldifferences that are not readily apparent in IVF.

Our previous studies had shown that the removal of the oocyte genomereduced developmental potential¹⁴. Tachibana and colleagues suggestedthat this may be because the removal of the oocyte genome compromisesthe meiotic state, condensation of somatic chromatin, thereby affectingembryonic development. However, we found that chromosome condensationoccurs at a similar frequency in enucleated and nucleated oocytes. Wealso found no difference in developmental potential if the somaticgenome was transferred 30 min before or after the removal of the oocytegenome (FIG. 11D). Instead, our data point to cell fusion as a cause fora compromised meiotic state observed after nuclear transfer. Fusion actsby compromising plasma membrane integrity, and can thereby lead to anincrease in intracellular calcium levels and failure to condense somaticchromatin (FIG.4). The use of low concentrations of fusogenic Sendaivirus and calcium-free media during cell fusion improved developmentalpotential and allowed derivation of stems from adult somatic cells (FIG.2A, FIG. 11C). Therefore, a likely interpretation of our previousobservations is that the retention of the oocyte genome promotesdevelopmental potential by compensating for inefficient reprogramming ofthe transferred somatic genome. Improvements in the nuclear transferprotocol remove this requirement of the oocyte genome for developmentafter nuclear transfer. Future studies should lead to a betterunderstanding of the molecular mechanisms how these technicalimprovements affect reprogramming and developmental outcome.

In summary, we have demonstrated the derivation of human ES cells fromadult somatic cells by nuclear transfer. The stem cells are pluripotentand could be differentiated into insulin producing beta cells, the celltype lost in patients with type 1 diabetes. Thus, somatic cell nucleartransfer may be a useful strategy to generate cells for therapeutic cellreplacement, a concept demonstrated in proof of principle experiments inimmunodeficient⁵ and in parkinsonian mice⁴. Though it is now possible toinduce stem cell formation by overexpression of transcription factors⁶,these cells often are differentiation defective', contain aberrantpatterns of cytosine methylation^(9,10,25) and hydroxymethylation⁸,somatic coding mutations²⁶, and show biallelic expression of imprintedgenes¹¹. Studies comparing isogenic nuclear transfer ES cells to iPScells generated from the same somatic cell cultures should enableevaluating the quality of cells generated by different methods ofreprogramming.

Materials and Methods Oocyte Donors

Oocytes were obtained as previously reported^(14,16). In brief, oocytedonors were recruited from an oocyte donation program. Potentialparticipants discussed the research with a physician and were offeredstudy participation. Upon choosing to donate for research, subjects gavesigned informed consent for the study protocol and the use of theiroocytes in nuclear transfer research. During a period of three years andfour months, 35 subjects donated a total of 512 mature MII oocytes,(average=14.63 oocytes /cycle, range=2-31 oocytes/cycle). 423 of theseoocytes were used for developmental analyses after somatic cell nucleartransfer reported here. 6 of the donors had one or more previouspregnancies, and 5 had previously donated for reproductive purposes.There were no repeat donations for this study.

Both GnRH antagonist (n=33) and GnRH agonist (n=2) protocols were used.The GnRH antagonist protocol was performed by administering dailysubcutaneous rFSH (recombinant follicle stimulating hormone) injectionsstarting on day 2 of the menstrual cycle and the addition of daily GnRHantagonist (Ganirelix) starting on day 6 of stimulation. Final oocytematuration was triggered after reaching a follicle size of 18 mm with 4mg GnRH agonist (Lupron) and 1000IU hCG (human chorionic gonadotropin,Novarel).

The GnRH agonist protocol was performed by administering daily GnRHagonist for at least 14 days followed by daily rFHS injections. Finalmaturation trigger was performed by administering 10,000IU hCG.

For both agonist and antagonist protocols, the dose of rFSH generallyconsisted of 2 ampules a day (range 1-3, average 2.49) and the time ofstimulation was 10 days (range 9-13 days, average 10.29). Initial dosewas established clinically based on subject age, baseline antralfollicle count, and anti-mullerian substance (AMH) level. Doseadjustments and the total days of stimulation were adjusted individuallybased on follicle number and size and serum estradiol (E2) levels.Hormones were administered by clinical staff, to ensure consistentapplication. At the time of oocyte retrival, venipuncture was performedfor DNA isolation. All human subjects' research was reviewed by aninstitutional review board and stem cell committees.

Oocyte Manipulations

Oocytes were enucleated using microtubule birefringence (Oosight) asdescribed previously by Noggle et al., 2011. Nuclear transfer wasperformed by fusion of somatic cells to oocytes using inactivated Sendaivirus diluted 1:10 to 1:20 in suspension medium. Fusion was confirmedvisually. 1-3 hours post fusion, oocytes were activated using 3 μMionomycin in Global total for 5 minutes at 37 degrees celsius, followedby culture in 10 μM puromycin, 2 mM 6-DMAP, as well as the histonedeacetylase (HDAC) inhibitors scriptaid and nch51 for 4 to 4.5 hours.Activated constructs were cultured in HDAC inhibitor for an additional10-16 hours, followed by culture in Global total only. Culture wasperformed in a MINC incubator fed with gas containing 6% CO2, 5% oxygenand 89% nitrogen. Calcium-free medium was used for some of themanipulations resulting in nuclear transfer ES cells.

For some of the experiments described above additional modificationswere introduced as described in Tachibana et al., 2013. Caffeine at a 1mM concentration was added to the incubation medium during enucleationand oocyte fusion. Oocytes were activated using 2-4 pulses of 50 μswidth at 2.7 kV/cm in mannitol containing fusion medium using an LF201pulser (Nepagene), followed by 4 hours of culture in 2 mM 6-DMAP.Activated constructs were cultured in Global total containing 10% FBSand TSA for 12 hours, followed by culture to the blastocysts stage inmedium containing 10% FBS.

The derivation of nuclear transfer ES cell lines from BJ fibroblasts atpassage 11 was performed using nuclear transfer 30 min prior to theremoval of the oocyte genome in the presence of 1 mM caffeine.Derivation of the adult cell NT-ES cell line was performed using somaticcells at passage 8, from a female subject with T1D (ID-1018, age ofonset 10y, age at study 32y). Oocytes were transported in GMOPSpluscontaining 1 mM caffeine. Upon completion of the manipulation, oocyteswere incubated for 45 min-1 h in calcium-free MCZB, then placed incalcium containing medium for 10 min prior to activation and culture asdescribed above. Oocyte activation was performed within 2 hours posttransfer using 4 electrical pulses, followed by incubation in mediumcontaining HDAC inhibitors scriptaid and nch51, in addition to 10 μg/mlpuromycin and 2 mM 6-DMAP for 4 hours, and thereafter, medium containingHDAC inhibitor for 15 hours. Culture was performed in a Heracell 150iincubator containing 5% CO2 at 37° C., in Global medium (IVFonlineLGGG-050) containing 10% fetal bovine serum. ES cell derivation wasconducted in medium containing DMEM/F12 supplemented with 10% KO-SR and10% FBS, Rock inhibitor Y-27632, thiazovivin, non-essential amino acids,10 ng/ml bFGF, Glutamax, and beta mercaptoethanol (all reagents fromLife Technologies). Upon attachment, trophectoderm cells were ablatedusing laser pulses using a Lykos laser (Hamilton Thorne). Outgrowthswere apparent within 10 days of plating, and were picked when coloniesreached a size of about 1 mm in diameter. Passaging was doneenzymatically using TrpLE (Life Technologies) and standard ES cellmedium (substituting FBS with KO-SR and DMEM/F12 with KO-DMEM) usingRock inhibitor Y-27632 for the first day after plating.

Karyotyping and Cell-Line Analysis

Karyotyping of human cell lines was done by Cell Line Genetics. Geneexpression analysis was performed using Illumina HumanHT-12 expressionBeadChip and analyzed using the Illumina BeadStudio software. Pancreaticdifferentiation was performed as described ²⁹. Datapoints in FIG. 2include comparisons to samples from GEO (GSE28024). Gene expressionanalysis of nuclear transfer ES cells and parental fibroblasts wasperformed using the Human Gene 1.0 ST microarray platform (Affymetrix)according to the manufacturer's protocol. Array data were analyzed usingRobust Multi-array Average (RMA) in Affymetrix Expression Console.Comparisons of gene expression levels and principle component analysisincluded additional published samples available on GEO. Neurons weredifferentiated for 34 days using a modified dual SMAD inhibitionprotocol³⁰. For beta cell differentiation, ES cells were dissociated intrypsin (Gibco), suspended in human ES medium containing 10 μM ROCKinhibitor (Y27632), plated at a density of 150,000 and 800,000 cells perwell in 48 and 12-well plates respectively and beta cell differentiationwas initiated 24 hours later. Detailed formulations of differentiationmedia were described previously³¹. Insulin secretion upon stimulationwith 30 mM KCl was measured by ELISA as previously described³¹.Embryonic bodies were generated in DMEM containing 10% FBS and allowedto grow for 3-4 weeks until analysis by immunocytochemistry. Forteratoma analysis, stem cells were injected subcutaneously into thedorsal flank of immunocompromised mice NOD.Cg-PrkdcscidIl2rg^(tmlWjl)/SzJ (Stock 005557), allowed to grow for 10-15 weeks andthen subjected to histological examination with hematoxylin and eosin(HE) staining.

Cytochalasin B (Sigma) was used for removal of the nuclear genome fromthe oocyte. GMOPSplus (Vitrolife, #10130) was used to transport oocytesand for manipulation of oocytes. Global (IVF online #LGGG-050) was usedto culture oocytes. A certified gas mixture of 5% oxygen, 6% carbondioxide, and 89% nitrogen (Techair T202250) was used for cell culture. Avitrification kit and a cryotop thawing kit (Kitazato) were used.Humagen micropipets were used for extraction of the oocyte nucleargenome. PCR reagents used for genotyping included: primers (IDT DNAtechnologies) and RedTaq polymerase (Sigma). Various chemicals (fromSigma) were used: Mannitol, MgSO4, BSA, HEPES buffer (for the generationof a fusion buffer). Ionomycin and puromycin were obtained from Sigma.Inactivated Sendai virus HVJ-E was obtained from GenomeOne, Cosmo Bio. ARoche genomic DNA isolation kit was used. A gene chip Human mapping 250KNsp Array and assay kit (Affymetrix #900767 and #900766) was used forsnp genotyping of donor somatic cell DNA. The following instruments wereused for all manipulations: a Nikon T2000-U microscope equipped with aNarishige micromanipulator, Oosight system, and a Tokai hit heatingplate. A portable incubator (INC-RB1, CryoLogic) was used to transportoocytes. Electrofusion was performed using an LF201 electrofusioninstrument (NepaGene). A Hamilton Thorne Laser system was also used.Additional details of the materials and methods used are providedelsewhere in this application and/or are well known in the art.

Whether nuclear transfer occurred before or after removal of the oocytegenome was inconsequential, and we also found no difference inefficiency of development to the blastocyst stage between adult orneonatal cells (FIG. 10D).

Calcium Imaging

The removal of the oocyte genome results in a karyoplast containingcytoplasm surrounded by a nuclear envelope. These were placed in a zonapellucida, and incubated for 30 min in fluo-4, using the fluo-4 directcalcium assay kit (Life Technologies). In brief, fluo-4 is added 1:1 toculture media. Karyoplasts were imaged both before and after exposure toinactivated Sendai virus using a Nikon TE200U microscope with an ET GFP(C92865, 96362) filter and a black and white camera and exposure time of200 ms.

Statistical Analysis

All oocytes on which experimentation has been performed are included inthis manuscript. Oocytes were randomly assigned to a specificexperimental condition. There was no blinding to group allocation. Tocalculate whether the differences in developmental potential betweendifferent experimental conditions were significant, we compared thetotal number of morulae and blastocysts between different conditions. Toreflect the greater developmental progression of a blastocyst versus amorula, each developmental stage was assigned a cell number: 15 formorula stage, 30 for a blastocyst. These numbers were chosen to reflectthe greater number of cells contained at each developmental stage(compacted morulas contain approximately 15 cells, blastocysts at least30 cells). Blastocysts giving rise to stem cell lines were not givengreater weight in this analysis, because blastocysts giving rise to stemcell lines did not necessarily have a greater cell number (SupplementaryFIG. 1). For each condition, the number of morulas was multiplied byfactor 15, the number of blastocysts by factor 30. Variables werecompared with Chi-square test, and the 95% confidence intervals usingGraphpad Prism v. 2.01 (Graphpad Software, USA).

Example 2

The transfer of somatic cell nuclei into oocytes can give rise to stemcells and cloned animals, but often development fails at early cleavagestages with an arrest in cell proliferation. We conducted a detailedanalysis of the mechanisms of developmental arrest after nucleartransfer into human and mouse oocytes and found that defects inchromosome segregation are frequent, arise prior to transcriptionalactivation of the genome, and required the transition through the firstembryonic interphase. Chromosome segregation errors involved theformation of bridges, acentric chromosome fragments, characteristic ofreplication-induced errors. Their frequency was greatly increased withthe state of differentiation of the donor nucleus, but reduced by theaddition of histone deacetylation inhibitors. These results demonstratethat the cell type specific duplication and segregation of the genome isa defining feature of a cellular state and a limiting factor for cellcycle progression during development. Here we report methods designed toovercome this limitation.

We have previously reported that human oocytes reprogram somatic cellsto a pluripotent state if the oocyte genome is not removed (Noggle etal., 2011). In the absence of the oocyte genome, nuclear transfer cellsarrested cell division at the cleavage stages and failed to activate anembryonic gene expression program. In contrast, when the oocyte genomewas retained, the transfer of somatic cell nuclei allowed the derivationof two stem cell lines containing a haploid oocyte genome and areprogrammed diploid somatic cell genome. These experiments demonstratedthe ability of human oocytes to reprogram a somatic cell to apluripotent state. To develop a protocol allowing the derivation ofdiploid stem cell lines, we sought to better understand how theretention of the oocyte genome facilitated pre-implantation development.The triploid nature of the stem cell lines derived suggested that theextrusion of the second polar body had occurred on the genome of theoocyte, but not the genome of the somatic cell. A possibleinterpretation of this asymmetry is that the oocyte spindle sequestersmost or all components necessary for spindle formation, and aretherefore not available to transferred somatic chromatin. They wouldalso be depleted with the removal of the oocyte genome at enucleation,which could affect the segregation of chromatin at embryonic mitosis,preventing normal development. To address these questions, weinvestigated spindle assembly and chromosome segregation after somaticcell nuclear transfer at both meiosis and mitosis.

Here we show that nuclear transfer embryos often arrest with karyotypeabnormalities and DNA damage. Spindle assembly and chromosomesegregation of somatic chromatin is normal at the second meioticdivision, but chromosome segregation errors are frequent duringembryonic mitoses. These errors are caused by replication of somaticchromatin in the embryonic cell, and precede the transcriptionalactivation of the genome. Their mechanisms of formation is not due to afailure of microtubules to attach and segregate somatic centromeres, butprimarily due to a failure to normally resolve sister chromatid cohesionat anaphase of mitosis. Our results show that differences in nuclearstructure and DNA replication between different cell types arefunctionally significant, acting as a barrier to experimentally inducedcell-type transitions. We propose that the cell-type specificduplication of the genome provides a mechanism tying cell cycleprogression to cell identity.

Mitotic, but not Meiotic, Segregation Errors after Human SCNT

We next sought to determine the timing and molecular mechanism of thesechromosomal abnormalities. We reasoned that if the removal of the oocytespindle removed essential components, then these defects should beapparent at the second meiotic division. We removed the oocytespindle-chromosome complex, and transferred the mitotic genome of asomatic cell that had been collected upon treatment with nocodazole andwere kept on ice prior to transfer (FIG. 12A). Remarkably, within 1-2hours post transfer, birefringent spindles were assembled in oocytes andchromosomes were aligned on the metaphase plate (FIG.12A, 12B, 12C).Upon artificial activation using a calcium ionophore and puromycin,16/19 oocytes segregated somatic cell chromatin into a polar body andformed a single pronucleus in the activated oocyte (FIG. 12F-I). Andlike upon segregation of oocyte chromatin, a midbody formed and thechromosome passenger complex component survivin localized to the midbody(FIG. 12E). To examine the accuracy of chromosome segregation, (pseudo-)polar bodies were biopsied and the karyotype determined using nucleotidepolymorphism arrays. The karyotype was balanced, and heterozygositypresent on all chromosomes, indicating that a diploid genome of 46sister chromatids was segregated into the polar body. Of 9 polar bodiesanalyzed, one contained a single karyotyic abnormality, an error ratecomparable to that seen upon transfer of an oocyte genome (Paull et al.,2013). Therefore, the oocyte has the ability to nucleate a spindle andaccurately segregate somatic chromatin even after removal of theoocyte's own metaphaseII spindle. However, when we analyzed thekaryotype of blastomeres using nucleotide arrays, we found a largenumber of karyotypic abnormalities (FIG. 12I-L). Of a total of 55blastomeres, 39 (71%) were abnormal (FIG. 12I, 12J). On average,blastomeres contained 3-4 abnormalities, and they occurred both aftertransfer of interphase or mitotic nuclei into enucleated oocytes (FIG.12K). In addition to numerical abnormalities, 87 of 138 blastomeres(63%) contained more than a single nucleus, significantly more oftenthan in IVF embryos (FIG. 12L). And of 30 nuclear transfer embryos, allbut one contained at least one multinucleated blastomere.

To directly observe the formation of chromosome abnormalities weanalyzed spindle assembly and chromosome segregation at the firstmitosis and in blastomeres. Though chromosomes could align on ametaphase plate in 3 of 4 embryos, two contained a chromosome that wasnot integrated into a spindle (FIG. 12M). The lack of a centromere (FIG.12M) suggested that not a defect in the spindle apparatus, but astructural deficiency of the chromosome was responsible for a lack ofspindle attachment. Upon entry into anaphase, we found anaphase bridges,chromosome fragments, lagging chromosomes and the dissociation ofsegregating chromosomes into several groups (FIG. 12N-P). Of 10 dividingcells, all of them contained at least one of these abnormalities.Therefore, the formation of chromosomal abnormalities could occur withinthe first cell cycle after somatic cell nuclear transfer. Abnormalchromosome segregation was also observed in mitotic blastomeres, withsome chromosomes failing to integrate into the metaphase plate (FIG.12Q). Such mitotic figures contained foci of phosphorylated γH2axstaining, evidence of DNA damage. Evidence of DNA damage was also seenin blastomeres at interphase, including foci of phosphorylated γH2ax andreplication protein A (RPA32) (FIG. 4R, 4S). These cells arrested atinterphase, with phosphorylation of Ser10 of histone H3, indicating thatthese cells progressed through most of S-phase, but failed to entermitosis. These results are consistent with our previous observation thatnuclear transfer embryos upregulate transcription of Gadd45 familymembers (Noggle et al., 2011), proteins induced following DNA damage andcapable of inducing cell cycle arrest at G2 (Wang et al., 1999). Thepresence of multiple nuclei in many of these blastomeres (FIG. 12S)indicated an abnormal prior mitosis. These results demonstrate thatchromosome segregation errors are not due to abnormalities in theembryonic spindle apparatus, nor due to the removal of the oocytespindle-chromosome complex, but arise during the first interphase upontransfer by an unknown mechanism.

Genomic Instability is Cell-Type and DNA Replication Dependent

To further investigate the mechanism of genomic instability aftersomatic cell nuclear transfer, we transferred genomes of somatic andembryonic mouse cells into enucleated mouse oocytes. As a control forour manipulations, we transplanted oocyte genomes from one MII oocyte toanother. We first quantified the number of abnormal anaphase figures inunmanipulated oocytes, including bridges, fragments, and mis-segregatingchromosomes. Abnormalities in parthenotes were few, or less than 5percent at anaphase of the second meiosis and the first mitosis (FIG.13A). The transfer of the oocyte genome (ONGT=oocyte nuclear genometransfer) from one oocyte to another did not increase the frequency ofthese abnormalities, neither at the second meiosis, nor at the firstmitosis. Cultured cells showed slightly higher formation of chromosomebridges and missegregating chromosomes than oocytes, or 6.3% forfibroblasts, and 8.2% for ES cells. Upon transfer of mitotic fibroblastor ES cell genomes into MII oocytes, the frequency of theseabnormalities remained unaltered, or 6% and 6.9%. However, at the firstmitosis, both ES cells and fibroblasts showed a significant increase inabnormally segregating chromosomes (FIG. 13A, 13C). Abnormal anaphasefigures were further increased when using interphase genomes of eithercumulus cells or T cells. These results demonstrate that the frequencyof abnormal chromosome segregation at mitosis is greatly dependent onthe developmental stage of the transferred genome (FIG. 13A).

As different cell types are known to differ in their gene expressionpattern, we first determined whether gene expression during the firstcell cycle was required for a normal chromosome segregation at the firstembryonic mitosis. The addition of the polymerasell inhibitor alphaamanitin to the culture medium did not significantly increase thefrequency of normal mitotic figures (FIG. 13A). As cell-type specificgene expression appeared to be irrelevant to chromosome segregation atthe first mitosis, we investigated whether other cell-type specificproperties of the genome were of importance. It appeared conceivablethat for normal chromosome segregation, the prior duplication of thegenome at interphase must also be normal. To distinguish whether thechromosome segregation we observed were due to abnormal spindleattachment to chromosomes or due to structural deficiencies that canarise during DNA replication, we stained anaphases with antibodiesrecognizing centromeres. All chromosomes (31/31) that failed tosegregate to the spindle poles lacked a centromere (FIG. 13D, 13E).Chromosome bridges contained a centromere mark that moved towards thespindle pole, but centromeres lagged behind normally segregatingchromosomes (8/8) (FIG. 13D). Lagging chromosomes were found to occur inpairs, protruding from each side of a group of anaphase chromosomes(FIG. 13F, 13G mouse). These chromosomes too contained a delayedcentromere, and probably formed bridges at an earlier time point ofanaphase. Though we cannot exclude that some bridges are successfullyresolved without damage to the genome, we found chromatin in the midbodyof 2-cell embryos at interphase (FIG. 13H), and evidence for DNA damagein interphase blastomeres (FIG. 13I). Nuclear transfer embryos were mostfrequently affected by chromosomes that did not incorporate into thespindle, followed by bridges and lagging chromosomes (FIG. 13K). Thesedefects are reminiscent of those caused by DNA replication stress insomatic cells (Chan et al., 2009).

To determine whether other parameters known to affect the developmentalefficiency after somatic cell nuclear transfer affected the fidelity ofsegregation at the first mitosis, we investigated the role of thenuclear structure. To preserve a somatic nuclear structure, weenucleated oocytes as before, but transferred somatic cell nuclei onlyafter activation and inhibition of meiotic kinases using 6-dmap. Duringartificial activation, kinase levels decrease, compromising the abilityto break down the nuclear envelope and induce chromosome condensation.When nuclei are transferred at prometaphase of mitosis, somaticchromatin condenses, allowing an efficient transition to a nucleus ofembryonic morphology, while nuclei transferred after activation retain asomatic morphology (Egli et al., 2011). We transferred somatic nucleiwithin the first hour post activation, and analyzed chromosomesegregation at the first mitosis. The large majority (90%) of dividingembryos were severely abnormal (FIG. 13L), most containing a string ofchromatin between the segregating spindle poles (FIG. 13M). Therefore,the reduction in kinase activities was detrimental to the ability ofoocytes to prepare somatic chromatin for progression through the firstcell cycle.

Most importantly, the addition of the histone deacetylation inhibitorscriptaid decreased the frequency of chromosome segregation errors (FIG.13L). Other epigenetic modifiers, including different histonedeacetylase inhibitors, as well as histone methylation inhibitors mayhave similar beneficial effects.

Discussion

Here we describe frequent chromosome segregation errors after somaticcell nuclear transfer into human oocytes. These defects are not due tothe depletion of spindle components removed with the oocyte chromatin,and they are not due to the transfer procedure itself. In addition, theyoccur prior to the major wave of embryonic genome activation in human(Braude et al., 1988) and are not caused by a lack of embryonictranscription. Using different donor cell types in mouse embryos, wefound a tight correlation of segregation errors with the state ofdifferentiation of the donor cell, which in turn correlates withdevelopmental potential. The nature of chromosome segregation errors,including bridges and acentric chromosomes, are reminiscent to thoseobserved after DNA replication stress (Burrell et al., 2013; Chan etal., 2009), and their frequency is altered by the presence of additionalnucleosides, compounds known to affect the activity of origins.Therefore, our results demonstrate that cell-type specific differencesin DNA replication are a functionally important barrier to cellreprogramming, and perhaps more generally, to cell type transitions.

Differences in gene expression are a defining feature of different celltypes. A large body of literature contributed to a better understandingof gene expression changes in development, differentiation andreprogramming. On the basis of this work, the mechanisms of cellreprogramming and development are often interpreted exclusively in thecontext of changes in gene expression (Hanna et al., 2010; Jaenisch andYoung, 2008). For instance, incomplete reactivation of embryonic genesis thought to be a major cause for the developmental failure of mouseclones (Boiani et al., 2002; Bortvin et al., 2003; Humpherys et al.,2002). Such emphasis on gene expression in cell-type reprogrammingimplies that progression through S-phase can occur independently of theregulators that determine cell identity. This model is at first sightattractive, because it confers the ability to progress through S-phaseto the many cell types arising during development and celldifferentiation, but also to abnormal cells, including tumor cells.However, the data presented here, and those by others, question thismodel. Nuclear transfer embryos with incomplete reprogramming arrestduring early development (Noggle et al., 2011), and only reprogrammedcells continue proliferation to yield ES cells equivalent to those offertilized embryos (Brambrink et al., 2006). Therefore, the ability ofcells to progress through S-phase and as a consequence through a normalM-phase depends on cell-type specific factors, tying cell proliferationto a specific cell identity.

DNA replication progresses in cell-type specific temporal patterns, anddiffers in even closely related cell types (Hiratani et al., 2010; Rybaet al., 2011). Upon reprogramming to induced pluripotent stem cells, orafter somatic cell nuclear transfer, DNA replication timing is changedfrom a somatic to an embryonic pattern (Hiratani et al., 2010; Shufaroet al., 2010). While Hiratani and colleagues used a genome-wide approachand discovered regions of replication timing that are difficult toreprogram, Shufaro and colleagues examined a small number ofdevelopmental genes that were efficiently reprogrammed. The recentfinding that oocytes remodel chromatin in a context-dependent manner,with efficient removal of DNA methylation at gene promoters, butinefficiently at LINE and LTR repeats (Chan et al., 2012), suggests thatthe reprogramming of DNA replication timing may also sometimes beincomplete.

Some of the factors regulating cell-type specific DNA replication may beidentical to those regulating cell-type specific gene expression. Bothprocesses depend on the three-dimensional organization of the nucleus.When a somatic cell nuclear structure is preserved in embryonic cells,normal progression through the cell cycle cannot occur. Similarly, whennuclear transfer is performed in mouse zygotes without remodeling of thesomatic nuclear structure, embryonic gene expression fails (Egli et al.,2011). As most proliferating cells undergo gene expression as well asDNA replication, it is challenging to distinguish effects of theexperimental manipulation on gene expression from effects on DNAreplication. For instance histone deacetylase inhibitors used toincrease reprogramming efficiency to iPS cells (Huangfu et al., 2008)are known to alter the activity of replication origins (Kemp et al.,2005) and gene expression. And c-myc, a protein used with Oct-4, Sox-2and Klf4 to generate iPS cells stimulates both transcription (Kato etal., 1990) and DNA replication (Dominguez-Sola et al., 2007). Nucleartransfer into oocytes provides a suitable model excluding geneexpression as a relevant factor, because the proteins and mRNA requiredfor normal cell cycle progression are provided maternally within theegg. We hypothesize that the cell type specificity of DNA replicationprovides a mechanism to control and limit cell proliferation dependingon cell state, in processes as diverse as aging, tumor formation,reprogramming, development and differentiation.

Immunocytochemistry

Oocytes and nuclear transfer cells were analyzed using the followingantibodies recognizing beta tubulin (Millipore 05-661), anti-centromere(15-235-0001 Antibodies Inc), phospho-histone H3 Ser10 (Millipore06-570), borealin (MBL Int Corp 147-3). Images were taken using a ZeissLSM710 confocal microscope, or a Zeiss LSMS Pascal microscope.Immunostaining of human stem cell lines was done using antibodies forOCT4 (09-0023, Stemgent), nanog (Cell Signaling Technologies D73G4),Tra1-81 (MAB4381 Millipore), and SSEA-4, anti-TRA1-60 (MAB4360;Millipore), PDX1 (R&D AF2419), Insulin (Millipore 05-1109), rabbitanti-AFP (A000829; DAKO) and rabbit anti-TUJ1 (T3952; Sigma-Aldrich).Hoechst33342 (Sigma) was used for the staining of DNA, secondaryantibodies are from LifeTechnologies. Cells were fixed in 2% PFA in PBScontaining 2.5% Triton-X100 at RT for about 10-15 min. Cells werewashed, blocked with FBS and incubated with primary antibodies. Imageswere taken using an Olympus IX71 epifluorescence microscope and anOlympus DP30 monochrome camera. Figures for publication were assembledin Adobe Illustrator.

Nuclear transfer methods in this Example are identical to thosedescribed in Example 1.

Example 3 Use of Metaphase Somatic Cells for Nuclear Transfer

Experiments were performed in which the nuclear genome was removed froman oocyte at the metaphase II stage to form an enucleated oocyte. Asomatic cell at mitosis was then transferred into/fused with the oocyteusing inactivated Sendai virus. The reconstructed oocyte was thencultured in the incubator (6% CO2, 5% O2, 89%N2, at 37° C.), for 2hours. The reconstructed oocyte comprising the somatic cell genome wasthen placed in an incubator and activated using a calcium ionophore at aconcentration of 2.5 μM in calcium free medium to induce a calcium pulseof a physiological amplitude. Upon activation, the egg was placed inmedium containing 10 μg/ml puromycin (a translation inhibitor). Themedium also contained 10 ng/ml of scriptaid—a histone deacetylationinhibitor. After 3.4-4 hours, the medium was changed to a mediumcontaining only 41 ng/ml scriptaid (but not puromycin) and maintainedfor 15 to 17 hours post activation. The scriptaid medium was thenremoved and embryos were cultured in Globaltotal medium. FIG.4A providesa schematic diagram of the protocol proceeding from enucleation of theoocyte, to fusion of the enucleated oocyte with a somatic cell inmitosis, and then spindle assembly of the transferred somatic cellgenome. FIG. 12B-12E show spindle assembly after nuclear transfer of asomatic cell genome. While some have claimed success using human cellsalso (see French et al. 2008), others have found that the development ofhuman oocytes after genome exchange arrests at late cleavage stages ifthe oocyte genome is removed (see Noggle et al. 2011). The noveltechniques described and exemplified herein allow efficientreprogramming and development to the blastocyst stage. Developmentalcompetence of nuclear transfer embryos to the blastocyst stage, asachieved here, is necessary for the derivation of pluripotent stem celllines.

Methods

Methods are identical to Example 1, with the exception of omitting6-dmap during oocyte activation. The somatic cell may be fused incalcium-free medium or incubated in BAPTA-AM prior to transfer to ensuremaintenance of the mitotic state.

REFERENCE LIST

Each of the references listed below, and all other references cited inthis patent application, are hereby incorporated by reference in theirentireties.

REFERENCES LISTED BY FIRST AUTHOR

Byrne, J. A., Pedersen, D. A., Clepper, L. L., Nelson, M., Sanger, W.G., Gokhale, S., Wolf, D. P., and Mitalipov, S. M. 2007. Producingprimate embryonic stem cells by somatic cell nuclear transfer. Nature450(7169): 497-502.

Chung, Y., Bishop, C. E., Treff, N. R., Walker, S. J., Sandler, V. M.,Becker, S., Klimanskaya, I., Wun, W. S., Dunn, R., Hall, R. M. et al.2009. Reprogramming of human somatic cells using human and animaloocytes. Cloning Stem Cells 11(2): 213-223.

French et al., 2008. Development of human cloned blastocysts followingsomatic cell nuclear transfer with adult fibroblasts. Stem Cells26(2):485-93.

Wakayama, T., Tabar, V., Rodriguez, I., Perry, A. C., Studer, L., andMombaerts, P. 2001. Differentiation of embryonic stem cell linesgenerated from adult somatic cells by nuclear transfer. Science292(5517): 740-743.

Wilmut, I., Schnieke, A. E., McWhir, J., Kind, A. J., and Campbell, K.H. 1997. Viable offspring derived from fetal and adult mammalian cells.Nature 385(6619): 810-813.

U.S. Patent Application Pub. No. US2012/0129620, by Egli et al. Boiani,M., Eckardt, S., Scholer, H. R. and McLaughlin, K. J. (2002). Oct4distribution and level in mouse clones: consequences for pluripotency.Genes Dev 16, 1209-19.

Bortvin, A., Eggan, K., Skaletsky, H., Akutsu, H., Berry, D. L.,Yanagimachi, R., Page, D. C. and Jaenisch, R. (2003). Incompletereactivation of Oct4-related genes in mouse embryos cloned from somaticnuclei. Development 130, 1673-80.

Brambrink, T., Hochedlinger, K., Bell, G. and Jaenisch, R. (2006). EScells derived from cloned and fertilized blastocysts aretranscriptionally and functionally indistinguishable. Proc Natl Acad SciU S A 103, 933-8.

Braude, P., Bolton, V. and Moore, S. (1988). Human gene expression firstoccurs between the four- and eight-cell stages of preimplantationdevelopment. Nature 332, 459-61.

Bui, H. T., Wakayama, S., Kishigami, S., Park, K. K., Kim, J. H., Thuan,N. V. and Wakayama, T. (2010). Effect of trichostatin A on chromatinremodeling, histone modifications, DNA replication, and transcriptionalactivity in cloned mouse embryos. Biol Reprod 83, 454-63.

Burrell, R. A., McClelland, S. E., Endesfelder, D., Groth, P., Weller,M. C., Shaikh, N., Domingo, E., Kanu, N., Dewhurst, S. M., Gronroos, E.et al. (2013). Replication stress links structural and numerical cancerchromosomal instability. Nature 494, 492-6.

Chan, K. L., Palmai-Pallag, T., Ying, S. and Hickson, I. D. (2009).Replication stress induces sister-chromatid bridging at fragile siteloci in mitosis. Nat Cell Biol 11, 753-60.

Chan, M. M., Smith, Z. D., Egli, D., Regev, A. and Meissner, A. (2012).Mouse ooplasm confers context-specific reprogramming capacity. Nat Genet44, 978-80.

Dominguez-Sola, D., Ying, C. Y., Grandori, C., Ruggiero, L., Chen, B.,Li, M., Galloway, D. A., Gu, W., Gautier, J. and Dalla-Favera, R.(2007). Non-transcriptional control of DNA replication by c-Myc. Nature448, 445-51.

Egli, D., Chen, A. E., Saphier, G., Ichida, J., Fitzgerald, C., Go, K.J., Acevedo, N., Patel, J., Baetscher, M., Kearns, W. G. et al. (2011).Reprogramming within hours following nuclear transfer into mouse but nothuman zygotes. Nat Commun 2, 488.

Hanna, J. H., Saha, K. and Jaenisch, R. (2010). Pluripotency andcellular reprogramming: facts, hypotheses, unresolved issues. Cell 143,508-25.

Hiratani, I., Ryba, T., Itoh, M., Rathjen, J., Kulik, M., Papp, B.,Fussner, E., Bazett-Jones, D. P., Plath, K., Dalton, S. et al. (2010).Genome-wide dynamics of replication timing revealed by in vitro modelsof mouse embryogenesis. Genome Res 20, 155-69.

Huangfu, D., Maehr, R., Guo, W., Eijkelenboom, A., Snitow, M., Chen, A.E. and Melton, D. A. (2008). Induction of pluripotent stem cells bydefined factors is greatly improved by small-molecule compounds. NatBiotechnol 26, 795-7.

Humpherys, D., Eggan, K., Akutsu, H., Friedman, A., Hochedlinger, K.,Yanagimachi, R., Lander, E. S., Golub, T. R. and Jaenisch, R. (2002).Abnormal gene expression in cloned mice derived from embryonic stem celland cumulus cell nuclei. Proc Natl Acad Sci U S A 99, 12889-94.

Jaenisch, R. and Young, R. (2008). Stem cells, the molecular circuitryof pluripotency and nuclear reprogramming. Cell 132, 567-82.

Kato, G. J., Barrett, J., Villa-Garcia, M. and Dang, C. V. (1990). Anamino-terminal c-myc domain required for neoplastic transformationactivates transcription. Mol Cell Biol 10, 5914-20.

Kemp, M. G., Ghosh, M., Liu, G. and Leffak, M. (2005). The histonedeacetylase inhibitor trichostatin A alters the pattern of DNAreplication origin activity in human cells. Nucleic Acids Res 33,325-36.

Kishigami, S., Mizutani, E., Ohta, H., Hikichi, T., Thuan, N. V.,Wakayama, S., Bui, H. T. and Wakayama, T. (2006). Significantimprovement of mouse cloning technique by treatment with trichostatin Aafter somatic nuclear transfer. Biochem Biophys Res Commun 340, 183-9.

Noggle, S., Fung, H. L., Gore, A., Martinez, H., Satriani, K. C.,Prosser, R., Oum, K., Paull, D., Druckenmiller, S., Freeby, M. et al.(2011). Human oocytes reprogram somatic cells to a pluripotent state.Nature 478, 70-5.

Paull, D., Emmanuele, V., Weiss, K. A., Treff, N., Stewart, L., Hua, H.,Zimmer, M., Kahler, D. J., Goland, R. S., Noggle, S. A. et al. (2013).Nuclear genome transfer in human oocytes eliminates mitochondrial DNAvariants. Nature 493, 632-7.

Robinton, D. A. and Daley, G. Q. (2012). The promise of inducedpluripotent stem cells in research and therapy. Nature 481, 295-305.

Ryba, T., Hiratani, I., Sasaki, T., Battaglia, D., Kulik, M., Zhang, J.,Dalton, S. and Gilbert, D. M. (2011). Replication timing: a fingerprintfor cell identity and pluripotency. PLoS Comput Biol 7, e1002225.

Rybouchkin, A., Kato, Y. and Tsunoda, Y. (2006). Role of histoneacetylation in reprogramming of somatic nuclei following nucleartransfer. Biol Reprod 74, 1083-9.

Schwartz, S. D., Hubschman, J. P., Heilwell, G., Franco-Cardenas, V.,Pan, C. K., Ostrick, R. M., Mickunas, E., Gay, R., Klimanskaya, I. andLanza, R. (2012). Embryonic stem cell trials for macular degeneration: apreliminary report. Lancet 379, 713-20.

Shufaro, Y., Lacham-Kaplan, O., Tzuberi, B. Z., McLaughlin, J.,Trounson, A., Cedar, H. and Reubinoff, B. E. (2010). Reprogramming ofDNA replication timing. Stem Cells 28, 443-9.

Tabar, V., Tomishima, M., Panagiotakos, G., Wakayama, S., Menon, J.,Chan, B., Mizutani, E., Al-Shamy, G., Ohta, H., Wakayama, T. et al.(2008). Therapeutic cloning in individual parkinsonian mice. Nat Med 14,379-81.

Tachibana, M., Amato, P., Sparman, M., Gutierrez, N. M., Tippner-Hedges,R., Ma, H., Kang, E., Fulati, A., Lee, H.-S., Sritanaudomchai, H. et al.(2013a). Human Embryonic Stem Cells Derived by Somatic Cell NuclearTransfer Cell.

Tachibana, M., Amato, P., Sparman, M., Gutierrez, N. M., Tippner-Hedges,R., Ma, H., Kang, E., Fulati, A., Lee, H. S., Sritanaudomchai, H. et al.(2013b). Human Embryonic Stem Cells Derived by Somatic Cell NuclearTransfer. Cell.

Wang, X. W., Zhan, Q., Coursen, J. D., Khan, M. A., Kontny, H. U., Yu,L., Hollander, M. C., O'Connor, P. M., Fornace, A. J., Jr. and Harris,C. C. (1999). GADD45 induction of a G2/M cell cycle checkpoint. ProcNatl Acad Sci U S A 96, 3706-11.

NUMERICAL LISTING OF REFERENCES

1. Gurdon, J.B., Elsdale, T.R., & Fischberg, M., Sexually matureindividuals of Xenopus laevis from the transplantation of single somaticnuclei. Nature 182 (4627), 64-65 (1958).

2. Noggle, S. et al., Human oocytes reprogram somatic cells to apluripotent state. Nature 478 (7367), 70-75 (2011).

3. Tachibana, M. et al., Human embryonic stem cells derived by somaticcell nuclear transfer. Cell 153 (6), 1228-1238 (2013).

4. Cyranoski, D., Verdict: Hwang's human stem cells were all fakes.Nature 439 (7073), 122-123 (2006).

5. Chung, Y. et al., Reprogramming of human somatic cells using humanand animal oocytes. Cloning Stem Cells 11 (2), 213-223 (2009).

6. Hall, V. J. et al., Developmental competence of human in vitro agedoocytes as host cells for nuclear transfer. Hum Reprod 22 (1), 52-62(2007).

7. Greggains, G. D. et al., Therapeutic potential of somatic cellnuclear transfer for degenerative disease caused by mitochondrial DNAmutations. Sci Rep 4, 3844 (2014).

8. Egli, D. et al., Reprogramming within hours following nucleartransfer into mouse but not human zygotes. Nat Commun 2, 488 (2011).

9. Stojkovic, M. et al., Derivation of a human blastocyst afterheterologous nuclear transfer to donated oocytes. Reprod Biomed Online11 (2), 226-231 (2005).

10. French, A. J. et al., Development of human cloned blastocystsfollowing somatic cell nuclear transfer with adult fibroblasts. StemCells 26 (2), 485-493 (2008).

11. Li, J. et al., Human embryos derived by somatic cell nucleartransfer using an alternative enucleation approach. Cloning Stem Cells11 (1), 39-50 (2009).

12. Fan, Y. et al., Derivation of cloned human blastocysts by histonedeacetylase inhibitor treatment after somatic cell nuclear transfer withbeta-thalassemia fibroblasts. Stem Cells Dev (2011).

13. Wakayama, T., Perry, A. C., Zuccotti, M., Johnson, K. R., &Yanagimachi, R., Full-term development of mice from enucleated oocytesinjected with cumulus cell nuclei. Nature 394 (6691), 369-374 (1998).

14. Liu, L., Oldenbourg, R., Trimarchi, J. R., & Keefe, D. L., Areliable, noninvasive technique for spindle imaging and enucleation ofmammalian oocytes. Nat Biotechnol 18 (2), 223-225 (2000).

15. Gassmann, R. et al., Borealin: a novel chromosomal passengerrequired for stability of the bipolar mitotic spindle. J Cell Biol 166(2), 179-191 (2004).

16. Kishigami, S. et al., Significant improvement of mouse cloningtechnique by treatment with trichostatin A after somatic nucleartransfer. Biochem Biophys Res Commun 340 (1), 183-189 (2006).

17. Rybouchkin, A., Kato, Y., & Tsunoda, Y., Role of histone acetylationin reprogramming of somatic nuclei following nuclear transfer. BiolReprod 74 (6), 1083-1089 (2006).

18. Paull, D. et al., Nuclear genome transfer in human oocyteseliminates mitochondrial DNA variants. Nature 493 (7434), 632-637(2013).

19. Braude, P., Bolton, V., & Moore, S., Human gene expression firstoccurs between the four- and eight-cell stages of preimplantationdevelopment. Nature 332 (6163), 459-461 (1988).

20. Kind, A. & Colman, A., Therapeutic cloning: needs and prospects.Semin Cell Dev Biol 10 (3), 279-286 (1999).

21. Rideout, W. M., 3rd, Hochedlinger, K., Kyba, M., Daley, G. Q., &Jaenisch, R., Correction of a genetic defect by nuclear transplantationand combined cell and gene therapy. Cell 109 (1), 17-27 (2002).

22. Tabar, V. et al., Therapeutic cloning in individual parkinsonianmice. Nat Med 14 (4), 379-381 (2008).

23. Klitzman, R. & Sauer, M. V., Payment of egg donors in stem cellresearch in the USA. Reprod Biomed Online 18 (5), 603-608 (2009).

24. Egli, D. et al., Impracticality of egg donor recruitment in theabsence of compensation. Cell Stem Cell 9 (4), 293-294 (2011).

25. Choudhary, M. et al., Egg sharing for research: a successful outcomefor patients and researchers. Cell Stem Cell 10 (3), 239-240 (2012).

26. Medicine, E.C.O.T.A.S.F.R., Financial compensation of oocyte donors.Fertil Steril 88 (2), 305-309 (2007).

27. Daley, G. Q. et al., Ethics. The ISSCR guidelines for humanembryonic stem cell research. Science 315 (5812), 603-604 (2007).

28. Tachibana, M., Sparman, M., & Mitalipov, S., Chromosome transfer inmature oocytes. Nat Protoc 5 (6), 1138-1147 (2010).

29. Hua, H. et al., iPSC-derived beta cells model diabetes due toglucokinase deficiency. J Clin Invest (2013).

30. Chambers, S. M. et al., Highly efficient neural conversion of humanES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27(3), 275-280 (2009).

31. Shang, L. et al., Beta cell dysfunction due to increased ER stressin a stem cell model of Wolfram syndrome. Diabetes (2013).

What is claimed is:
 1. A method for producing a diploid human nucleartransfer embryo capable of developing into a blastocyst containing aninner cell mass, the method comprising: a. obtaining a diploid nucleargenome from a postnatal human somatic cell, b. obtaining an enucleatedmature human oocyte, c. transferring the diploid nuclear genome into theenucleated mature human oocyte to form a reconstructed oocyte, whereinthe transferring is performed in a medium that is calcium-free, and/orcontains a calcium chelator, and/or contains a phosphatase inhbitor, d.subsequently contacting the reconstructed oocyte with a calciumionophore, an inhibitor of translation, and an inhibitor meiotickinases, to activate the reconstructed oocyte and promote entry intointerphase, and e. contacting the reconstructed oocyte with a histonedeacetylase inhibitor and/or a histone methylation inhibitor, therebyproducing a diploid human nuclear transfer embryo capable of developinginto a blastocyst containing an inner cell mass.
 2. The method of claim1, wherein the inhibitor of translation is puromycin and the inhibitorof meiotic kinases is 6-DMAP.
 3. The method of claim 2 wherein thepuromycin is used at approximately 10 μM and wherein the 6-DMAP is usedat approximately 2 mM.
 4. The method of claim 1, further comprisingculturing the diploid human nuclear transfer embryo in the presence offetal bovine serum (FBS) until it reaches the blastocyst stage.
 5. Themethod of claim 1, wherein step (c) comprises using a fusogenic agent.6. The method of claim 5, wherein the fusogenic agent is used at theminimum concentration sufficient to induce cell fusion.
 7. The method ofclaim 5, wherein the fusogenic agent is an inactivated Sendai virusSendai virus HVJ-E.
 8. The method of claim 1, wherein the histonedeacetylase inhibitor is selected from the group consisting ofScriptaid, Nch51 and trichostatin.
 9. The method of claim 1, wherein thereconstructed oocyte, or embryo derived from the reconstructed oocyte,is contacted with the histone deacetylase inhibitor(s) for approximately14 to approximately 21 hours post activation, or until just prior to thefirst mitosis.